Human ataxin-1-like polypeptide IMX97018

ABSTRACT

This invention relates to IMX97018, a new members of the human ataxin-1-like polypeptide family, methods of making such polypeptides, and to methods of using them to diagnose and treat neurological conditions and to identify compounds that alter IMX97018 polypeptide activities.

This application is a divisional of U.S. application Ser. No.10/997,561, filed Nov. 23, 2004, now allowed, which is a divisional ofU.S. application Ser. No. 10/207,706, filed Jul. 26, 2002, now U.S. Pat.No. 6,887,687, which claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application Ser. No. 60/309,056, filed Jul. 30, 2001. Theentire disclosures of these applications are relied upon andincorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to IMX97018, a new ataxin-1-like humanpolypeptide, and to methods of making and using IMX97018 polypeptides.

BACKGROUND OF THE INVENTION

Ataxin-1 is one of a group of polypeptides implicated inspinocerebrellar ataxia (SCA) conditions, also called autosomal dominantcerebellar ataxias (ADCAs). SCA disorders are heritable autosomaldominant neurodegenerative conditions commonly featuring progressiveataxia, which is irregularity of muscle action due to failure of musclecoordination. Examples of other symptoms typically shown by patientsdiagnosed with SCA are paralysis of the ocular muscles (ophthalmoplegia)and loss of articulation of speech (dysarthria), as associated with SCA2and SCA7; degeneration and loss of types of brain cells, as in loss ofcerebellar Purkinje cells in SCA1 for example; and dementia, asassociated with SCA2 and SCA6.

Several of the SCA disorders are characterized by genetic anticipation,which is the tendency of certain diseases to appear at earlier onsetages and/or with increased severity in each successive generation. Inmany cases, genetic anticipation has been shown to have a biologicalbasis in the expansion in length of a stretch of triplet repeats thatencode a particular amino acid. SCA1 shows genetic anticipation and isassociated with expansions in the size of a polyglutamine tract inataxin-1 polypeptide encoded by repeated CAG codons (Matilla et al.,1993, Presymptomatic analysis of spinocerebellar ataxia type 1 (SCA1)via the expansion of the SCA1 CAG-repeat in a large pedigree displayinganticipation and parental male bias, Hum Molec Genet 2: 2123-2128).Genetic anticipation has also been observed in families afflicted withseveral other of the SCA disorders such as SCA2, SCA3, SCA5, SCA6, SCA7,SCA8, SCA10, and in addition to the ataxin-1 gene of SCA1, expansions ofCAG repeats have been found in alleles of those SCA genes that have beencharacterized to date: the SCA2/ataxin-2, SCA3/MJD1, SCA6/CACNA1A, andSCA7/ataxin-7 genes. Therefore, all of the SCA genes that have beenstudied at the molecular level indicate that expansions of CAG repeatsare correlated with the genetic anticipation observed in thecorresponding SCA disorder. While family history evidence has also beenpresented for genetic anticipation in SCA4 (Flanigan et al., 1996, Am JHum Genet 59: 392-399), the SCA4 gene has not yet been identified andcharacterized.

Ataxin-1, -2, and -7, ataxin-3/MJD1, and CACNA1A polypeptides aredetected in the cytoplasm of many types of neural cells, with the levelsof expression varying from cell type to cell type, and with overlappingbut non-identical patterns of expression displayed by these differentpolypeptides. The formation of nuclear inclusion bodies immunoreactivefor these SCA-related polypeptides is positively correlated with thelength of the polyglutamine tracts in the polypeptides. Interactions ofthese polypeptides with several different types of binding partners havebeen reported, and these interactions are believed to contribute indifferent ways to development of the SCA disease condition. For example,ataxin-1 polypeptide has been found to associate with cerebellarleucine-rich acidic nuclear protein (LANP) in the nuclear matrix ofPurkinje cells, the primary site of the pathological effects of SCA1(Matilla et al., 1997, Nature 389: 974-978). Association with nuclearproteins is thought to alter the conformation of ataxin-3/MJD1polypeptide, exposing the polyglutamine tract (Perez et al., 1999, HumMol Genet 8: 2377-2385). Certain SCA-related polypeptides have beenreported to have RNA-binding activity, either as part of the SCA-relatedpolypeptide itself, or by binding to a polypeptide with RNA-bindingactivity. For example, ataxin-1 polypeptide binds RNA in vitro, with theRNA-binding capability inversely proportional to the length of thepolyglutamine tract (Yue et al., 2001, Hum Mol Genet 10: 25-30), andataxin-2 interacts with ataxin-2 binding protein 1 (A2BP1), apolypeptide containing RNA-binding motifs (Shibata et al., 2000, Hum MolGenet 9: 1303-1313). Interestingly, in cells containing ataxin-1 with anexpanded glutamine tract, down-regulation of particular neuronal genesis postulated to be an early step in SCA1 pathogenesis (Lin et al.,2000, Nat Neurosci 3: 157-163). In addition, there is evidence thatpolyglutamine tracts tend to self-associate, sequestering polypeptidescontaining them in nuclear inclusions and possibly trapping otherpolypeptides required for cell viability, such as CREB-binding protein(CBP) (McCampbell et al., 2000, Hum Mol Genet 9: 2197-2202). Onepossible outcome of expression of polyglutamine-containing polypeptidesin neural cells is cell death through a non-apoptotic mechanism (Evertet al., 1999, Hum Mol Genet 8: 1169-1176).

In order to develop more effective treatments for spinocerebellarconditions and diseases, such as SCA1 and SCA4, information is neededabout previously unidentified or uncharacterized SCA-relatedpolypeptides.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery of a new humanataxin-1-like polypeptide, IMX97018.

The invention provides an isolated polypeptide consisting of, consistingessentially of, or more preferably, comprising an amino acid sequenceselected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:2;    -   (b) an amino acid sequence selected from the group consisting        of: amino acid 542 through amino acid 579 of SEQ ID NO:2, and        amino acid 464 through amino acid 583 of SEQ ID NO:2;    -   (c) an amino acid sequence selected from the group consisting        of: amino acids 185 through 195 of SEQ ID NO:2, amino acids 213        through 223 of SEQ ID NO:2, amino acids 190 through 218 of SEQ        ID NO:2, and amino acids 185 through 223 of SEQ ID NO:2;    -   (d) a fragment of the amino acid sequences of any of (a)-(c)        comprising at least 20 contiguous amino acids;    -   (e) a fragment of the amino acid sequences of any of (a)-(c)        comprising at least 30 contiguous amino acids;    -   (f) a fragment of the amino acid sequences of any of (a)-(c)        having IMX97018 polypeptide activity;    -   (g) a fragment of the amino acid sequences of any of (a)-(c)        comprising AXH domain amino acid sequences;    -   (h) amino acid sequences comprising at least 20 amino acids and        sharing amino acid identity with the amino acid sequences of any        of (a)-(g), wherein the percent amino acid identity is selected        from the group consisting of: at least 75%, at least 80%, at        least 85%, at least 90%, at least 95%, at least 97.5%, at least        99%, and at least 99.5%;    -   (i) an amino acid sequence of (h), wherein a polypeptide        comprising said amino acid sequence of (h) binds to an antibody        that also binds to a polypeptide comprising an amino acid        sequence of any of (a)-(g); and    -   (j) an amino acid sequence of (h) or (i) having IMX97018        polypeptide activity.

Other aspects of the invention are isolated nucleic acids encodingpolypeptides of the invention, with a preferred embodiment being anisolated nucleic acid consisting of, or more preferably, comprising anucleotide sequence selected from the group consisting of:

-   -   (a) SEQ ID NO:1; and    -   (b) an allelic variant of (a).

The invention also provides an isolated genomic nucleic acidcorresponding to the nucleic acids of the invention.

Other aspects of the invention are isolated nucleic acids encodingpolypeptides of the invention, and isolated nucleic acids, preferablyhaving a length of at least 15 nucleotides, and preferably at least 50%of the length of SEQ ID NO:1, that hybridize under conditions ofmoderate stringency to the nucleic acids encoding polypeptides of theinvention. In preferred embodiments of the invention, such nucleic acidsencode a polypeptide having IMX97018 polypeptide activity, or comprise anucleotide sequence that shares nucleotide sequence identity with thenucleotide sequences of the nucleic acids of the invention, wherein thepercent nucleotide sequence identity is selected from the groupconsisting of: at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 97.5%, at least 99%, and at least99.5%.

Further provided by the invention are expression vectors and recombinanthost cells comprising at least one nucleic acid of the invention, andpreferred recombinant host cells wherein said nucleic acid is integratedinto the host cell genome.

Also provided is a process for producing a polypeptide encoded by thenucleic acids of the invention, comprising culturing a recombinant hostcell under conditions promoting expression of said polypeptide, whereinthe recombinant host cell comprises at least one nucleic acid of theinvention. A preferred process provided by the invention furthercomprises purifying said polypeptide. In another aspect of theinvention, the polypeptide produced by said process is provided.

Further aspects of the invention are isolated antibodies that bind tothe polypeptides of the invention, preferably monoclonal antibodies,also preferably humanized antibodies or humanized antibodies, andpreferably wherein the antibody inhibits the activity of saidpolypeptides.

The invention additionally provides a method of designing an inhibitorof the polypeptides of the invention, the method comprising the steps ofdetermining the three-dimensional structure of any such polypeptide,analyzing the three-dimensional structure for the likely binding sitesof substrates, synthesizing a molecule that incorporates a predictedreactive site, and determining the polypeptide-inhibiting activity ofthe molecule. In a further aspect of the invention, a method is providedfor identifying compounds that alter IMX97018 polypeptide activitycomprising

-   -   (a) mixing a test compound with a polypeptide of the invention;        and    -   (b) determining whether the test compound alters the IMX97018        polypeptide activity of said polypeptide.

In another aspect of the invention, a method is provided identifyingcompounds that inhibit the binding activity of IMX97018 polypeptidescomprising

-   -   (a) mixing a test compound with a polypeptide of the invention        and a binding partner of said polypeptide; and    -   (b) determining whether the test compound inhibits the binding        activity of said polypeptide.        In preferred embodiments, the binding partner is a nuclear        polypeptide; more preferably, the binding partner is a        leucine-rich polypeptide, and still more preferably, the binding        partner is a LANP polypeptide.

Further provided by the invention is a method for decreasingSCA-promoting activity of IMX97018polyQ polypeptides, comprisingproviding at least one antagonist of the polypeptides of the invention;with a preferred embodiment of the method further comprising decreasingsaid activities in a patient by administering at least one antagonist ofthe polypeptides of the invention, and with a further preferredembodiment wherein the antagonist is an antisense molecule that inhibitsthe activity of any of said polypeptides, and with a most preferredembodiment wherein the antagonist specifically inhibits the activity ofpolyglutamine-containing forms of said polypeptides.

An additional aspect of the invention is a method for promoting celldeath comprising providing at least one compound selected from the groupconsisting of an IMX97018polyQ polypeptide and agonists of saidpolypeptides. Further provided by the invention is a method forinhibiting cell death activity comprising providing at least oneantagonist of an IMX97018polyQ polypeptide, for example wherein the celldeath activity is inhibited in neuronal cells.

The invention additionally provides a method for treating a oncologiccondition comprising administering at least one compound selected fromthe group consisting of an IMX9701 polypeptide and agonists of saidpolypeptide. In additional aspects of the invention, the oncologiccondition is selected from the group consisting of brain tumors, glioma,glioblastoma, astrocytoma, oligodendroglioma, ependymoma, ganglioglioma,medulloblastoma, neuroectodermal tumors, and pilocytic astrocytoma.

Also provided by the invention is a method for treating a neurologicalcondition comprising administering at least one compound selected fromthe group consisting of an IMX97018polyQ polypeptide and agonists ofsaid polypeptides, wherein the conditional is characterized by excessneurological activity. In addition, the invention provides a method fortreating a neurological condition comprising administering an antagonistan IMX97018polyQvpolypeptide. In further aspects of the invention, theneurological condition is selected from the group consisting ofdementia, including AIDS-related dementia and Alzheimer's.

In a further aspect of the invention, a method is provided for treatinga neuromuscular condition comprising administering an antagonist of anIMX97018polyQ polypeptide, for example wherein the neuromuscularcondition is ataxia.

In other aspects of the invention, a method is provided for treating aneurological condition comprising administering an antagonist of thepolypeptide of the invention; with a preferred embodiment wherein theneurological condition is SCA4.

A further embodiment of the invention provides a use for antagonists ofthe polypeptides of the invention in the preparation of a medicament fortreating a neurological condition; with a preferred embodiment whereinthe neurological condition is SCA4.

An additional aspect of the invention provides methods for mapping anddiagnosing genetic disorders linked to human chromosome 16, wherein thedisorder is preferably SCA4.

DETAILED DESCRIPTION OF THE INVENTION

Similarities of IMX97018 Structure to Ataxin-1 Polypeptides

We have identified IMX97018, a new ataxin-1-like polypeptide havingstructural features characteristic of mammalian ataxin-1 polypeptides;the amino acid sequence of an IMX97018 polypeptide is provided in SEQ IDNO:2, and an alignment showing the sequence similarities betweenIMX97018 and other ataxin-I polypeptides is presented in Table 1 inExample 1 below. Collectively, the set of polypeptides comprisingIMX97018 and the ataxin-1 polypeptides presented in Table 1, along withataxin-1 homologues from other species, are referred to as‘ataxin-1-like’ polypeptides. The ataxin-1-like polypeptides shown inTable 1 display a high degree of similarity to each other, with themammalian ataxin-1 polypeptides extremely similar to each other, andIMX97018 polypeptide sharing about 39% amino acid identity with humanataxin-1 and the mammalian ataxin-1 homologues.

The typical structural elements common to ataxin-1 polypeptides includean AXH domain, an ataxin-1 self-association domain, and an RNA-bindingdomain, and in some forms of ataxin-1 polypeptides, a polyglutaminetract resulting from expansion of CAG triplet repeats in the codingsequence (see Table 1 below). The AXH domain has been identified as adomain of 120 amino acids (SEQ ID NO:6) common to ataxin-1 polypeptidesfrom several species, and also some HMG-box-containing polypeptides (HMGbox containing protein 1 [Homo sapiens], GenBank AAB71862; HMG-boxcontaining protein 1 [Homo sapiens], GenBank.NP_(—)036389; and HMG-boxcontaining protein 1 [Rattus norvegicus], GenBank NP 037353;interestingly, HMG-box-containing polypeptides have been implicated inregulation of transcription initiation). Table 1 in Example 1 shows thelocation of the AXH domain within ataxin-1 and IMX97018 polypeptides,from amino acid 464 through amino acid 583 of SEQ ID NO:2, with aparticularly strong match between the AXH domain and the IMX97018polypeptide from amino acid 542 through amino acid 579 of SEQ ID NO:2. Aregion within the ataxin-1 polypeptide sufficient for self-associationin a yeast two-hybrid assay system is present at approximately aminoacids 495 through 605 of human ataxin-1 (SEQ ID NO:3) (Burright et al.,1997, Hum Molec Genet 6: 513-518), which corresponds to amino acids 431through 499 of IMX97018 polypeptide (SEQ ID NO:2). This ataxin-1self-association region is distinct from expanded polyglutamine tractsthat are also implicated in self-association of ataxin-1-likepolypeptides. A region of ataxin-1 polypeptide required for RNA-bindingactivity extends from amino acid 541 through amino acid 767 of SEQ IDNO:4 (Yue et al., 2001, Hum Mol Genet 10: 25-30); amino acids 444through 640 of IMX97018 polypeptide (SEQ ID NO:2) align with thisportion of ataxin-1. The portion of IMX97018 polypeptide that shows thegreatest degree of similarity to the ataxin-1 self-association regionare approximately amino acids 465 through 499 of SEQ ID NO:2, and theportion of IMX97018 polypeptide that shows the greatest degree ofsimilarity to the ataxin-1 RNA-binding region are approximately aminoacids 465 through 590 of SEQ ID NO:2; these portions of IMX97018polypeptide are also those that approximately correspond to the AXHdomain (amino acid 464 through amino acid 583 of SEQ ID NO:2). The humanataxin-1 polypeptide amino acid sequence shown as SEQ ID NO:3 has apolyglutamine tract of 28 Gln residues with two interspersed Hisresidues from amino acid 197 to amino acid 226 of SEQ ID NO:3. Themurine and rat ataxin-1 amino acid sequences shown as SEQ ID NO:4 andSEQ ID NO:5 have only two Gln residues at the position corresponding tothe polyglutamine tract in human ataxin-1. While IMX97018 does not havea polyglutamine tract, it does have two glutamine residues encoded byCAG codons in the region corresponding to the polyglutamine tract ofhuman ataxin-1: amino acid 190 and amino acid 218 of SEQ ID NO:2. Theregion of SEQ ID NO:2 corresponding to the polyglutamine tract of humanataxin-1 therefore preferably includes at least one of these Glnresidues, or amino acids 185 through 195 of SEQ ID NO:2, amino acids 213through 223 of SEQ ID NO:2, amino acids 190 through 218 of SEQ ID NO:2,or amino acids 185 through 223 of SEQ ID NO:2. IMX97018 polypeptides ofthe invention include isolated naturally occurring polypeptides havingpolyglutamine tracts, and IMX97018 polypeptides produced so as toinclude a polyglutamine tract; such polyglutamine-containing IMX97018polypeptides are referred to as ‘IMX97018polyQ polypeptides’ herein.Preferably, such polyglutamine tracts are greater than 30 contiguousglutamine and/or histidine residues in length; such polyglutamine tractshaving more than 30 residues are referred to as expanded polyglutaminetracts. More preferably, such polyglutamine tracts are 50 or more (or 60or more, or 70 or more, or 80 or more, or 90 or more, or 100 or more, or150 or more, or 200 or more, or 300 or more) contiguous glutamine and/orhistidine residues in length. In IMX97018 polypeptides havingpolyglutamine tracts, the polyglutamine sequence is preferably insertedin the polyglutamine region of the IMX97018 polypeptide, i.e. at aposition between amino acids 185 through 223 of SEQ ID NO:2; and morepreferably between amino acids 185 through 195, amino acids 213 through223, or amino acids 190 through 218 of SEQ ID NO:2; and most preferablyat the glutamine residue at amino acid 190 or at amino acid 218 of SEQID NO:2.

Therefore, IMX97018 polypeptide has an overall structure consistent withother ataxin-1-like polypeptides. The skilled artisan will recognizethat the boundaries of the regions of IMX97018 polypeptides describedabove are approximate and that the precise boundaries of such domains,as for example the boundaries of the region corresponding to the humanataxin-1 polyglutamine tract, can also differ from member to memberwithin ataxin-1-like polypeptide family.

Biological Activities and Functions of IMX97018 Polypeptides

Typical biological activities or functions associated with ataxin-1 andataxin-1-like polypeptides include RNA-binding activity andself-association activity. For ataxin-1 and ataxin-1-like polypeptidescomprising expanded polyglutamine tracts, activities associated withsuch polypeptides include promoting the formation of nuclear inclusions;binding to nuclear polypeptides, for example to leucine-richpolypeptides such as LANP; down-regulating certain genes expressed inneural tissue; and promoting cell death, preferably through anon-apoptotic mechanism. The RNA-binding and self-association activitiesof ataxin-1 are associated with a portion of the C-terminal portion ofthe polypeptide which includes the AXH domain. Thus, for uses requiringRNA-binding activity, preferred IMX97018 polypeptides include thosehaving the RNA-binding region, that is, amino acids 465 through 590 ofSEQ ID NO:2 and more preferably amino acids 465 through 583 of SEQ IDNO:2. For uses requiring self-association activity, preferred IMX97018polypeptides include those having the self-association region, that is,amino acids 465 through 499 of SEQ ID NO:2. Preferred IMX97018polypeptides further include oligomers or fusion polypeptides comprisingat least one AXH domain portion of one or more IMX97018 polypeptides,and fragments of any of these polypeptides that have RNA-bindingactivity or self-association activity. The RNA-binding activity ofIMX97018 polypeptides can be determined, for example, in an assay thatmeasures the amount of radiolabeled IMX97018 polypeptide that binds toagarose beads coated with a ribohomopolymer such as poly(rG). IMX97018polypeptides (those without expanded polyglutamine tracts) havingRNA-binding activity preferably have at least 25% (more preferably, atleast 50%, and most preferably, at least 75%) of the RNA-bindingactivity of ataxin-1 having a 30-glutamine tract as measured in Yue etal., 2001, Hum Mol Genet 10: 25-30 (see FIG. 4). Ataxin-1-likepolypeptides with expanded polyglutamine tracts (i.e. having more than30 contiguous glutamine and/or histidine residues) are expected to haveless RNA-binding activity in such assays than ataxin-1 having a30-glutamine tract. The self-association activity of IMX97018polypeptides can be determined, for example, in a yeast two-hybrid assayin which both the DNA-binding fusion protein and the activation-domainfusion protein contain IMX97018 polypeptides comprising theself-association region. IMX97018 polypeptides having self-associationactivity preferably have at least 25% (more preferably, at least 50%,and most preferably, at least 75%) of the self-association activity ofataxin-1 having a 30-glutamine tract as measured in a quantitativeassay, such as the ONPG assay of Burright et al., 1997, Hum Molec Genet6: 513-518 (see Table 2 on p. 514 of Burright et al.).

IMX97018 polypeptides, such as those having expanded polyglutaminetracts, which have nuclear inclusion formation activity promote theformation of IMX97018-immunoreactive nuclear aggregates. Additionalactivities of IMX97018 polypeptides, such as those having expandedpolyglutamine tracts, include binding to leucine-rich nuclearpolypeptides; down-regulation of certain genes expressed in neuraltissue; and promotion of cell death. These activities are associatedwith the region of IMX97018 polypeptides corresponding to thepolyglutamine tract of human ataxin-1 polypeptide. Thus, for usesrequiring nuclear inclusion formation activity, leucine-rich nuclearpolypeptide binding activity, neural gene down-regulation activity, orpromotion of cell death activity, preferred IMX97018 polypeptidesinclude those having the polyglutamine region and exhibiting one or moreof the above biological activities; further preferred IMX97018polypeptides include those having the an expanded polyglutamine tractand exhibiting one or more of the above biological activities. PreferredIMX97018 polypeptides further include oligomers or fusion polypeptidescomprising at least one polyglutamine region of one or more IMX97018polypeptides, and fragments of any of these polypeptides that have oneor more of the above polyglutamine-associated activities. The nuclearinclusion formation activity and the cell death promotion activity ofIMX97018 polypeptides can be determined, for example, throughimmunohistochemical and microscopic imaging techniques, such as confocalmicroscopy as used by Skinner et al. (1998, Nature 389: 971-974), orelectron microscopy as used by Evert et al. (1999, Hum Mol Genet 8:1169-1176). IMX97018 polypeptides having nuclear inclusion formationactivity preferably promote nuclear inclusion formation in at least 10%(more preferably, at least 25%, and most preferably, at least 50%) ofthe percentage of cells having nuclear inclusions, when such cellsexpress human ataxin-1 with a polyglutamine tract of 82 residues, whenmeasured as in Skinner et al., 1998, Nature 389: 971-974. IMX97018polypeptides having cell death promotion activity preferably promotecell death in at least 10% (more preferably, at least 25%, and mostpreferably, at least 50%) of the percentage of necrotic cells, when suchcells express human ataxin-1 with a polyglutamine tract of 70 residues,when measured as in Evert et al., 1999, Hum Mol Genet 8: 1169-1176. Theneural gene down-regulation activity of IMX97018 polypeptides can bedetermined, for example, through assays such as Northern blots of RNAfrom neural cells expressing IMX97018 polypeptides, or quantitative PCRfrom such cells, where the probe(s) or primers are specific forparticular genes expressed in neural cells. IMX97018 polypeptides havingneural gene down-regulation activity preferably have at least 10% (morepreferably, at least 25%, and most preferably, at least 50%) of theneural gene down-regulation activity of human ataxin-1 with apolyglutamine tract of 82 residues, as measured by quantification ofNorthern blot band intensity.

The term “IMX97018 polypeptide activity,” as used herein, includes anyone or more of the following: RNA-binding activity, self-associationactivity, nuclear inclusion formation promoting activity, leucine-richnuclear protein binding activity, neural gene down-regulation activity,and cell death promoting activity, as well as the ex vivo and in vivoactivities of IMX97018 family polypeptides. The degree to whichindividual members of the IMX97018 polypeptide family and fragments andother derivatives of these polypeptides exhibit these activities can bedetermined by standard assay methods, particularly assays such as thosereferred to above. Exemplary assays are disclosed herein; those of skillin the art will appreciate that other, similar types of assays can beused to measure IMX97018 family biological activities.

An aspect of the biological activity of IMX97018 polypeptides is theability of members of this polypeptide family to bind particular bindingpartners such as nuclear polypeptides, particularly leucine-rich nuclearpolypeptides, and most particularly LANP polypeptides, with this bindingactivity associated with the region of IMX97018 polypeptidecorresponding to the polyglutamine tract of ataxin-1, and in anotherembodiment, with the polyglutamine tract in IMX97018 polypeptides havingsuch a polyglutamine tract. The term “binding partner,” as used herein,includes ligands, receptors, substrates, antibodies, other IMX97018polypeptides, the same IMX97018 polypeptide (in the case of homotypicinteractions), and any other molecule that interacts with an IMX97018polypeptide through contact or proximity between particular portions ofthe binding partner and the IMX97018 polypeptide. Because thepolyglutamine region or tract of IMX97018 polypeptides binds toleucine-rich polypeptides, the polyglutamine region or tract whenexpressed as a separate fragment from the rest of an IMX97018polypeptide, or as a soluble polypeptide, fused for example to animmunoglobulin Fc domain, is expected to disrupt the binding of IMX97018polypeptides to their binding partners. By binding to one or morebinding partners, the separate polyglutamine region or tract polypeptidelikely prevents binding by native IMX97018 polypeptide(s), and so actsin a dominant negative fashion to inhibit the biological activitiesmediated via binding of IMX97018 polypeptides to binding partners suchas leucine-rich nuclear polypeptides. Particularly suitable assays todetect or measure the binding between IMX97018 polypeptides and theirbinding partners are yeast two-hybrid assays and in vitro bindingassays.

IMX97018 polypeptides are involved in neurological diseases orconditions, that share as a common feature disruption of neural cellmorphology and neural cell death in their etiology. More specifically,the following neurological conditions are those that are known or arelikely to involve the biological activities of IMX97018 polypeptides:spinocerebellar ataxia; particularly spinocerebellar ataxia type 4(SCA4). Blocking or inhibiting the interactions between members of theIMX97018 polypeptide family and their substrates, ligands, receptors,binding partners, and or other interacting polypeptides is an aspect ofthe invention and provides methods for treating or ameliorating thesediseases and conditions through the use of inhibitors of IMX97018polypeptide activity. Examples of such inhibitors or antagonists aredescribed in more detail below. For certain conditions involving toolittle IMX97018 polypeptide activity, methods of treating orameliorating these conditions comprise increasing the amount or activityof IMX97018 polypeptides by providing isolated IMX97018 polypeptides oractive fragments or fusion polypeptides thereof, or by providingcompounds (agonists) that activate endogenous or exogenous IMX97018polypeptides.

Additional uses for IMX97018 polypeptides include diagnostic reagentsfor diseases linked to human chromosome 16 and/or neurological disorderssuch as ataxic conditions, and research reagents for investigation ofpolyglutamine-containing polypeptides.

IMX97018 Polypeptides

An IMX97018 polypeptide is a polypeptide that shares a sufficient degreeof amino acid identity or similarity to the IMX97018 polypeptide of SEQID NO:2 to (A) be identified by those of skill in the art as apolypeptide likely to share particular structural domains and/or (B)have biological activities in common with the UMX97018 polypeptide ofSEQ ID NO:2 and/or (C) bind to antibodies that also specifically bind toother IMX97018 polypeptides. IMX97018 polypeptides can be isolated fromnaturally occurring sources, or have the same structure as naturallyoccurring IMX97018 polypeptides, or can be produced to have structuresthat differ from naturally occurring IMX97018 polypeptides. Polypeptidesderived from any IMX97018 polypeptide by any type of alteration (forexample, but not limited to, insertions, deletions, or substitutions ofamino acids; changes in the state of glycosylation of the polypeptide;refolding or isomerization to change its three-dimensional structure orself-association state; and changes to its association with otherpolypeptides or molecules) are also IMX97018 polypeptides. Therefore,the polypeptides provided by the invention include polypeptidescharacterized by amino acid sequences similar to those of the IMX97018polypeptides described herein, but into which modifications arenaturally provided or deliberately engineered. A polypeptide that sharesbiological activities in common with IMX97018 polypeptides is apolypeptide having IMX97018 polypeptide activity. Examples of biologicalactivities exhibited by IMX97018 polypeptides include, withoutlimitation, RNA-binding activity, self-association activity, nuclearinclusion formation promoting activity, leucine-rich nuclear proteinbinding activity, neural gene down-regulation activity, and cell deathpromoting activity.

The present invention provides both full-length and mature forms ofIMX97018 polypeptides. Full-length polypeptides are those having thecomplete primary amino acid sequence of the polypeptide as initiallytranslated. The amino acid sequences of full-length polypeptides can beobtained, for example, by translation of the complete open reading frame(“ORF”) of a cDNA molecule. Several full-length polypeptides can beencoded by a single genetic locus if multiple mRNA forms are producedfrom that locus by alternative splicing or by the use of multipletranslation initiation sites. The “mature form” of a polypeptide refersto a polypeptide that has undergone post-translational processing stepssuch as cleavage of the signal sequence or proteolytic cleavage toremove a prodomain. Multiple mature forms of a particular full-lengthpolypeptide may be produced, for example by cleavage of the signalsequence at multiple sites, or by differential regulation of proteasesthat cleave the polypeptide. The mature form(s) of such polypeptide canbe obtained by expression, in a suitable mammalian cell or other hostcell, of a nucleic acid molecule that encodes the full-lengthpolypeptide. The sequence of the mature form of the polypeptide may alsobe determinable from the amino acid sequence of the full-length form,through identification of signal sequences or protease cleavage sites.The IMX97018 polypeptides of the invention also include those thatresult from post-transcriptional or post-translational processing eventssuch as alternate mRNA processing which can yield a truncated butbiologically active polypeptide, for example, a naturally occurringsoluble form of the polypeptide. Also encompassed within the inventionare variations attributable to proteolysis such as differences in the N-or C-termini upon expression in different types of host cells, due toproteolytic removal of one or more terminal amino acids from thepolypeptide (generally from 1-5 terminal amino acids).

The invention further includes IMX97018 polypeptides with or withoutassociated native-pattern glycosylation. Polypeptides expressed in yeastor mammalian expression systems (e.g., COS-1 or CHO cells) can besimilar to or significantly different from a native polypeptide inmolecular weight and glycosylation pattern, depending upon the choice ofexpression system. Expression of polypeptides of the invention inbacterial expression systems, such as E. coli, provides non-glycosylatedmolecules. Further, a given preparation can include multipledifferentially glycosylated species of the polypeptide. Glycosyl groupscan be removed through conventional methods, in particular thoseutilizing glycopeptidase. In general, glycosylated polypeptides of theinvention can be incubated with a molar excess of glycopeptidase(Boehringer Mannheim).

Species homologues of IMX97018 polypeptides and of nucleic acidsencoding them are also provided by the present invention. As usedherein, a “species homologue” is a polypeptide or nucleic acid with adifferent species of origin from that of a given polypeptide or nucleicacid, but with significant sequence similarity to the given polypeptideor nucleic acid, as determined by those of skill in the art. Specieshomologues can be isolated and identified by making suitable probes orprimers from polynucleotides encoding the amino acid sequences providedherein and screening a suitable nucleic acid source from the desiredspecies. The invention also encompasses allelic variants of IMX97018polypeptides and nucleic acids encoding them; that is,naturally-occurring alternative forms of such polypeptides and nucleicacids in which differences in amino acid or nucleotide sequence areattributable to genetic polymorphism (allelic variation amongindividuals within a population).

Fragments of the IMX97018 polypeptides of the present invention areencompassed by the present invention and can be in linear form orcyclized using known methods, for example, as described in Saragovi etal., Bio/Technology 10, 773-778 (1992) and in McDowell et al., J. Amer.Chem. Soc. 114 9245-9253 (1992). Polypeptides and polypeptide fragmentsof the present invention, and nucleic acids encoding them, includepolypeptides and nucleic acids with amino acid or nucleotide sequencelengths that are at least 25% (more preferably at least 50%, or at least60%, or at least 70%, and most preferably at least 80%) of the length ofan IMX97018 polypeptide and have at least 60% sequence identity (morepreferably at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97.5%, or at least 99%, and mostpreferably at least 99.5%) with that IMX97018 polypeptide or encodingnucleic acid, where sequence identity is determined by comparing theamino acid sequences of the polypeptides when aligned so as to maximizeoverlap and identity while minimizing sequence gaps. Also included inthe present invention are polypeptides and polypeptide fragments, andnucleic acids encoding them, that contain or encode a segment preferablycomprising at least 8, or at least 10, or preferably at least 15, ormore preferably at least 20, or still more preferably at least 30, ormost preferably at least 40 contiguous amino acids. Such polypeptidesand polypeptide fragments may also contain a segment that shares atleast 70% sequence identity (more preferably at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%,or at least 99%, and most preferably at least 99.5%) with any suchsegment of any IMX97018 polypeptide, where sequence identity isdetermined by comparing the amino acid sequences of the polypeptideswhen aligned so as to maximize overlap and identity while minimizingsequence gaps. The percent identity of two amino acid or two nucleicacid sequences can be determined by visual inspection and mathematicalcalculation, or more preferably, the comparison is done by comparingsequence information using a computer program. An exemplary, preferredcomputer program is the Genetics Computer Group (GCG; Madison, Wis.)Wisconsin package version 10.0 program, ‘GAP’ (Devereux et al., 1984,Nucl. Acids Res. 12: 387).

The preferred default parameters for the ‘GAP’ program includes: (1) TheGCG implementation of a unary comparison matrix (containing a value of 1for identities and 0 for non-identities) for nucleotides, and theweighted amino acid comparison matrix of Gribskov and Burgess, Nucl.Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds.,Atlas of Polypeptide Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358, 1979; or other comparable comparisonmatrices; (2) a penalty of 30 for each gap and an additional penalty of1 for each symbol in each gap for amino acid sequences, or penalty of 50for each gap and an additional penalty of 3 for each symbol in each gapfor nucleotide sequences; (3) no penalty for end gaps; and (4) nomaximum penalty for long gaps. Other programs used by those skilled inthe art of sequence comparison can also be used, such as, for example,the BLASTN program version 2.0.9, available for use via the NationalLibrary of Medicine website www.ncbi.nlm.nih.gov/gorf/wblast2.cgi, orthe UW-BLAST 2.0 algorithm. Standard default parameter settings forUW-BLAST 2.0 are described at the following Internet site:sapiens.wust1.edu/blast/blast/#Features. In addition, the BLASTalgorithm uses the BLOSUM62 amino acid scoring matrix, and optionalparameters that can be used are as follows: (A) inclusion of a filter tomask segments of the query sequence that have low compositionalcomplexity (as determined by the SEG program of Wootton and Federhen(Computers and Chemistry, 1993); also see Wootton and Federhen, 1996,Analysis of compositionally biased regions in sequence databases,Methods Enzymol. 266: 554-71) or segments consisting ofshort-periodicity internal repeats (as determined by the XNU program ofClaverie and States (Computers and Chemistry, 1993)), and (B) astatistical significance threshold for reporting matches againstdatabase sequences, or E-score (the expected probability of matchesbeing found merely by chance, according to the stochastic model ofKarlin and Altschul (1990); if the statistical significance ascribed toa match is greater than this E-score threshold, the match will not bereported); preferred E-score threshold values are 0.5, or in order ofincreasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, 1e-5,1e-10, 1e-15, 1e-20, 1e-25, 1e-30, 1e-40, 1e-50, 1e-75, or 1e-100.

The present invention also provides for soluble forms of IMX97018polypeptides comprising certain fragments or domains of thesepolypeptides, and particularly those comprising the AXH domain, thepolyglutamine region or tract, or one or more fragments of thesedomains. Soluble polypeptides are polypeptides that are capable of beingsecreted from the cells in which they are expressed. Soluble IMX97018polypeptides include those forms of IMX97018 polypeptide that arecapable of being secreted from a cell, such as those to which a signalpeptide has been fused to the N-terminal end, and preferably those thatretain IMX97018 polypeptide activity. Soluble IMX97018 polypeptidesfurther include oligomers or fusion polypeptides, and fragments of anyof these polypeptides that have IMX97018 polypeptide activity. Asecreted soluble polypeptide can be identified (and distinguished fromits non-soluble counterparts) by separating intact cells which expressthe desired polypeptide from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired polypeptide. The presence of the desired polypeptide inthe medium indicates that the polypeptide was secreted from the cellsand thus is a soluble form of the polypeptide. The use of soluble formsof IMX97018 polypeptides is advantageous for many applications.Purification of the polypeptides from recombinant host cells isfacilitated, since the soluble polypeptides are secreted from the cells.Moreover, soluble polypeptides are generally more suitable forparenteral administration and for many enzymatic procedures.

In another aspect of the invention, preferred polypeptides comprisevarious combinations of IMX97018 polypeptide domains, such as the AXHdomain and the polyglutamine region or tract. Accordingly, polypeptidesof the present invention and nucleic acids encoding them include thosecomprising or encoding two or more copies of a domain such as the AXHdomain domain, two or more copies of a domain such as the polyglutamineregion or tract, or at least one copy of each domain, and these domainscan be presented in any order within such polypeptides.

Further modifications in the peptide or DNA sequences can be made bythose skilled in the art using known techniques. Modifications ofinterest in the polypeptide sequences can include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid. For example, one or more of the cysteine residues can be deletedor replaced with another amino acid to alter the conformation of themolecule, an alteration which may involve preventing formation ofincorrect intramolecular disulfide bridges upon folding or renaturation.Techniques for such alteration, substitution, replacement, insertion ordeletion are well known to those skilled in the art (see, e.g., U.S.Pat. No. 4,518,584). As another example, N-glycosylation sites in thepolypeptide extracellular domain can be modified to precludeglycosylation, allowing expression of a reduced carbohydrate analog inmammalian and yeast expression systems. N-glycosylation sites ineukaryotic polypeptides are characterized by an amino acid tripletAsn-X—Y, wherein X is any amino acid except Pro and Y is Ser or Thr.Appropriate substitutions, additions, or deletions to the nucleotidesequence encoding these triplets will result in prevention of attachmentof carbohydrate residues at the Asn side chain. Alteration of a singlenucleotide, chosen so that Asn is replaced by a different amino acid,for example, is sufficient to inactivate an N-glycosylation site.Alternatively, the Ser or Thr can by replaced with another amino acid,such as Ala. Known procedures for inactivating N-glycosylation sites inpolypeptides include those described in U.S. Pat. No. 5,071,972 and EP276,846. Additional variants within the scope of the invention includepolypeptides that can be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives can be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein. Preferably, such alteration, substitution, replacement,insertion or deletion retains the desired activity of the polypeptide ora substantial equivalent thereof. One example is a variant that bindswith essentially the same binding affinity as does the native form.Binding affinity can be measured by conventional procedures, e.g., asdescribed in U.S. Pat. No. 5,512,457 and as set forth herein.

Other derivatives include covalent or aggregative conjugates of thepolypeptides with other polypeptides or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.Examples of fusion polypeptides are discussed below in connection witholigomers. Further, fusion polypeptides can comprise peptides added tofacilitate purification and identification. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.One such peptide is the FLAG® peptide, which is highly antigenic andprovides an epitope reversibly bound by a specific monoclonal antibody,enabling rapid assay and facile purification of expressed recombinantpolypeptide. A murine hybridoma designated 4E11 produces a monoclonalantibody that binds the FLAG® peptide in the presence of certaindivalent metal cations, as described in U.S. Pat. No. 5,011,912. The4E11 hybridoma cell line has been deposited with the American TypeCulture Collection under accession no. HB 9259. Monoclonal antibodiesthat bind the FLAG® peptide are available from Eastman Kodak Co.,Scientific Imaging Systems Division, New Haven, Conn.

Encompassed by the invention are oligomers or fusion polypeptides thatcontain an IMX97018 polypeptide, one or more fragments of IMX97018polypeptides, or any of the derivative or variant forms of 11MX97018polypeptides as disclosed herein. In particular embodiments, theoligomers comprise soluble IMX97018 polypeptides. Oligomers can be inthe form of covalently linked or non-covalently-linked multimers,including dimers, trimers, or higher oligomers. In one aspect of theinvention, the oligomers maintain the binding ability of the polypeptidecomponents and provide therefor, bivalent, trivalent, etc., bindingsites. In an alternative embodiment the invention is directed tooligomers comprising multiple IMX97018 polypeptides joined via covalentor non-covalent interactions between peptide moieties fused to thepolypeptides, such peptides having the property of promotingoligomerization. Leucine zippers and certain polypeptides derived fromantibodies are among the peptides that can promote oligomerization ofthe polypeptides attached thereto, as described in more detail below.

In embodiments where variants of the IMX97018 polypeptides areconstructed to include a membrane-spanning domain, they will form a TypeI membrane polypeptide.

Immunoglobulin-based Oligomers. The polypeptides of the invention orfragments thereof can be fused to molecules such as immunoglobulins formany purposes, including increasing the valency of polypeptide bindingsites. For example, fragments of an IMX97018 polypeptide can be fuseddirectly or through linker sequences to the Fc portion of animmunoglobulin. For a bivalent form of the polypeptide, such a fusioncould be to the Fc portion of an IgG molecule. Other immunoglobulinisotypes can also be used to generate such fusions. For example, apolypeptide-IgM fusion would generate a decavalent form of thepolypeptide of the invention. The term “Fc polypeptide” as used hereinincludes native and mutein forms of polypeptides made up of the Fcregion of an antibody comprising any or all of the CH domains of the Fcregion. Truncated forms of such polypeptides containing the hinge regionthat promotes dimerization are also included. Preferred Fc polypeptidescomprise an Fc polypeptide derived from a human IgG1 antibody. As onealternative, an oligomer is prepared using polypeptides derived fromimmunoglobulins. Preparation of fusion polypeptides comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:677,1990); and Hollenbaugh and Aruffo (“Construction of ImmunoglobulinFusion Polypeptides”, in Current Protocols in Immunology, Suppl. 4,pages 10.19.1-10.19.11, 1992). Methods for preparation and use ofimmunoglobulin-based oligomers are well known in the art. One embodimentof the present invention is directed to a dimer comprising two fusionpolypeptides created by fusing a polypeptide of the invention to an Fcpolypeptide derived from an antibody. A gene fusion encoding thepolypeptide/Fc fusion polypeptide is inserted into an appropriateexpression vector. Polypeptide/Fc fusion polypeptides are expressed inhost cells transformed with the recombinant expression vector, andallowed to assemble much like antibody molecules, whereupon interchaindisulfide bonds form between the Fc moieties to yield divalentmolecules. One suitable Fc polypeptide, described in PCT application WO93/10151, is a single chain polypeptide extending from the N-terminalhinge region to the native C-terminus of the Fc region of a human IgG1antibody. Another useful Fc polypeptide is the Fc mutein described inU.S. Pat. No. 5,457,035 and in Baum et al., (EMBO J. 13:3992-4001,1994). The amino acid sequence of this mutein is identical to that ofthe native Fc sequence presented in WO 93/10151, except that amino acid19 has been changed from Leu to Ala, amino acid 20 has been changed fromLeu to Glu, and amino acid 22 has been changed from Gly to Ala. Themutein exhibits reduced affinity for Fc receptors. The above-describedfusion polypeptides comprising Fc moieties (and oligomers formedtherefrom) offer the advantage of facile purification by affinitychromatography over Polypeptide A or Polypeptide G columns. In otherembodiments, the polypeptides of the invention can be substituted forthe variable portion of an antibody heavy or light chain. If fusionpolypeptides are made with both heavy and light chains of an antibody,it is possible to form an oligomer with as many as four IMX97018extracellular regions.

Peptide-linker Based Oligomers. Alternatively, the oligomer is a fusionpolypeptide comprising multiple IMX97018 polypeptides, with or withoutpeptide linkers (spacer peptides). Among the suitable peptide linkersare those described in U.S. Pat. Nos. 4,751,180 and 4,935,233. A DNAsequence encoding a desired peptide linker can be inserted between, andin the same reading frame as, the DNA sequences of the invention, usingany suitable conventional technique. For example, a chemicallysynthesized oligonucleotide encoding the linker can be ligated betweenthe sequences. In particular embodiments, a fusion polypeptide comprisesfrom two to four soluble IMX97018 polypeptides, separated by peptidelinkers. Suitable peptide linkers, their combination with otherpolypeptides, and their use are well known by those skilled in the art.

Leucine-Ziplers. Another method for preparing the oligomers of theinvention involves use of a leucine zipper. Leucine zipper domains arepeptides that promote oligomerization of the polypeptides in which theyare found. Leucine zippers were originally identified in severalDNA-binding polypeptides (Landschulz et al., Science 240:1759, 1988),and have since been found in a variety of different polypeptides. Amongthe known leucine zippers are naturally occurring peptides andderivatives thereof that dimerize or trimerize. The zipper domain (alsoreferred to herein as an oligomerizing, or oligomer-forming, domain)comprises a repetitive heptad repeat, often with four or five leucineresidues interspersed with other amino acids. Use of leucine zippers andpreparation of oligomers using leucine zippers are well known in theart.

Other fragments and derivatives of the sequences of polypeptides whichwould be expected to retain polypeptide activity in whole or in part andmay thus be useful for screening or other immunological methodologiescan also be made by those skilled in the art given the disclosuresherein. Such modifications are believed to be encompassed by the presentinvention.

Nucleic Acids Encoding IMX97018 Polypeptides

Encompassed within the invention are nucleic acids encoding IMX97018polypeptides, such as SEQ ID NO:1, which encodes the IMX97018polypeptide of SEQ ID NO:2. These nucleic acids can be identified inseveral ways, including isolation of genomic or cDNA molecules from asuitable source. Nucleotide sequences corresponding to the amino acidsequences described herein, to be used as probes or primers for theisolation of nucleic acids or as query sequences for database searches,can be obtained by “back-translation” from the amino acid sequences, orby identification of regions of amino acid identity with polypeptidesfor which the coding DNA sequence has been identified. The well-knownpolymerase chain reaction (PCR) procedure can be employed to isolate andamplify a DNA sequence encoding an IMX97018 polypeptide or a desiredcombination of IMX97018 polypeptide fragments. Oligonucleotides thatdefine the desired termini of the combination of DNA fragments areemployed as 5′ and 3′ primers. The oligonucleotides can additionallycontain recognition sites for restriction endonucleases, to facilitateinsertion of the amplified combination of DNA fragments into anexpression vector. PCR techniques are described in Saiki et al., Science239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., AcademicPress, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guideto Methods and Applications, Innis et. al., eds., Academic Press, Inc.(1990).

Nucleic acid molecules of the invention include DNA and RNA in bothsingle-stranded and double-stranded form, as well as the correspondingcomplementary sequences. DNA includes, for example, cDNA, genomic DNA,chemically synthesized DNA, DNA amplified by PCR, and combinationsthereof. The nucleic acid molecules of the invention include full-lengthgenes or cDNA molecules as well as a combination of fragments thereof.The nucleic acids of the invention are preferentially derived from humansources, but the invention includes those derived from non-humanspecies, as well.

An “isolated nucleic acid” is a nucleic acid that has been separatedfrom adjacent genetic sequences present in the genome of the organismfrom which the nucleic acid was isolated, in the case of nucleic acidsisolated from naturally-occurring sources. In the case of nucleic acidssynthesized enzymatically from a template or chemically, such as PCRproducts, cDNA molecules, or oligonucleotides for example, it isunderstood that the nucleic acids resulting from such processes areisolated nucleic acids. An isolated nucleic acid molecule refers to anucleic acid molecule in the form of a separate fragment or as acomponent of a larger nucleic acid construct. In one preferredembodiment, the nucleic acids are substantially free from contaminatingendogenous material. The nucleic acid molecule has preferably beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

“An isolated nucleic acid consisting essentially of a nucleotidesequence” means that the nucleic acid may have, in addition to saidnucleotide sequence, additional material covalently linked to either orboth ends of the nucleic acid molecule, said additional materialpreferably between 1 and 100,000 additional nucleotides covalentlylinked to either end, each end, or both ends of the nucleic acidmolecule, and more preferably between 1 and 10,000 additionalnucleotides covalently linked to either end, each end, or both ends ofthe nucleic acid molecule, and most preferably between 10 and 1,000additional nucleotides covalently linked to either end, each end, orboth ends of the nucleic acid molecule. An isolated nucleic acidconsisting essentially of a nucleotide sequence may be an expressionvector or other construct comprising said nucleotide sequence. “Anisolated nucleic acid consisting essentially of a nucleotide sequence”further is meant to exclude isolated human chromosomes or isolatedcontigs such as GenBank accession number AC009127.

The present invention also includes nucleic acids that hybridize undermoderately stringent conditions, and more preferably highly stringentconditions, to nucleic acids encoding IMX97018 polypeptides describedherein. The basic parameters affecting the choice of hybridizationconditions and guidance for devising suitable conditions are set forthby Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., chapters 9 and 11; and Current Protocols in MolecularBiology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections2.10 and 6.3-6.4), and can be readily determined by those havingordinary skill in the art based on, for example, the length and/or basecomposition of the DNA. One way of achieving moderately stringentconditions involves the use of a prewashing solution containing 5×SSC,0.5% SDS, 1.0 m⁻¹ M EDTA (pH 8.0), hybridization buffer of about 50%formamide, 6×SSC, and a hybridization temperature of about 55 degrees C.(or other similar hybridization solutions, such as one containing about50% formamide, with a hybridization temperature of about 42 degrees C.),and washing conditions of about 60 degrees C., in 0.5×SSC, 0.1% SDS.Generally, highly stringent conditions are defined as hybridizationconditions as above, but with washing at approximately 68 degrees C.,0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH.sub.2 PO.sub.4,and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15MNaCl and 15 mM sodium citrate) in the hybridization and wash buffers;washes are performed for 15 minutes after hybridization is complete. Itshould be understood that the wash temperature and wash saltconcentration can be adjusted as necessary to achieve a desired degreeof stringency by applying the basic principles that govern hybridizationreactions and duplex stability, as known to those skilled in the art anddescribed further below (see, e.g., Sambrook et al., 1989). Whenhybridizing a nucleic acid to a target nucleic acid of unknown sequence,the hybrid length is assumed to be that of the hybridizing nucleic acid.When nucleic acids of known sequence are hybridized, the hybrid lengthcan be determined by aligning the sequences of the nucleic acids andidentifying the region or regions of optimal sequence complementarity.The hybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5 to 10.degrees C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(degrees C.)=2(# of A+T bases)+4(# of #G+C bases). For hybrids above 18base pairs in length, Tm (degrees C.)=81.5+16.6(log₁₀[Na⁺])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na⁺] isthe concentration of sodium ions in the hybridization buffer ([Na⁺] for1×SSC=0.165M). Preferably, each such hybridizing nucleic acid has alength that is at least 15 nucleotides (or more preferably at least 18nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, orat least 30 nucleotides, or at least 40 nucleotides, or most preferablyat least 50 nucleotides), or at least 25% (more preferably at least 50%,or at least 60%, or at least 70%, and most preferably at least 80%) ofthe length of the nucleic acid of the present invention to which ithybridizes, and has at least 60% sequence identity (more preferably atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97.5%, or at least 99%, and most preferably at least99.5%) with the nucleic acid of the present invention to which ithybridizes, where sequence identity is determined by comparing thesequences of the hybridizing nucleic acids when aligned so as tomaximize overlap and identity while minimizing sequence gaps asdescribed in more detail above.

The present invention also provides genes corresponding to the nucleicacid sequences disclosed herein. “Corresponding genes” or “correspondinggenomic nucleic acids” are the regions of the genome that aretranscribed to produce the mRNAs from which cDNA nucleic acid sequencesare derived and can include contiguous regions of the genome necessaryfor the regulated expression of such genes. Corresponding genes cantherefore include but are not limited to coding sequences, 5′ and 3′untranslated regions, alternatively spliced exons, introns, promoters,enhancers, and silencer or suppressor elements. Corresponding genomicnucleic acids can include 10000 basepairs (more preferably, 5000basepairs, still more preferably, 2500 basepairs, and most preferably,1000 basepairs) of genomic nucleic acid sequence upstream of the firstnucleotide of the genomic sequence corresponding to the initiation codonof the IMX97018 coding sequence, and 10000 basepairs (more preferably,5000 basepairs, still more preferably, 2500 basepairs, and mostpreferably, 1000 basepairs) of genomic nucleic acid sequence downstreamof the last nucleotide of the genomic sequence corresponding to thetermination codon of the IMX97018 coding sequence. The correspondinggenes or genomic nucleic acids can be isolated in accordance with knownmethods using the sequence information disclosed herein. Such methodsinclude the preparation of probes or primers from the disclosed sequenceinformation for identification and/or amplification of genes inappropriate genomic libraries or other sources of genomic materials. An“isolated gene” or “an isolated genomic nucleic acid” is a genomicnucleic acid that has been separated from the adjacent genomic sequencespresent in the genome of the organism from which the genomic nucleicacid was isolated.

Methods for Making and Purifying IMX97018 Polypeptides

Methods for making IMX97018 polypeptides are described below.Expression, isolation, and purification of the polypeptides andfragments of the invention can be accomplished by any suitabletechnique, including but not limited to the following methods. Theisolated nucleic acid of the invention can be operably linked to anexpression control sequence such as the pDC409 vector (Giri et al.,1990, EMBO J., 13: 2821) or the derivative pDC412 vector (Wiley et al.,1995, Immunity 3: 673). The pDC400 series vectors are useful fortransient mammalian expression systems, such as CV-1 or 293 cells.Alternatively, the isolated nucleic acid of the invention can be linkedto expression vectors such as pDC312, pDC316, or pDC317 vectors. ThepDC300 series vectors all contain the SV40 origin of replication, theCMV promoter, the adenovirus tripartite leader, and the SV40 polyA andtermination signals, and are useful for stable mammalian expressionsystems, such as CHO cells or their derivatives. Other expressioncontrol sequences and cloning technologies can also be used to producethe polypeptide recombinantly, such as the pMT2 or pED expressionvectors (Kaufman et al., 1991, Nucleic Acids Res. 19: 4485-4490; andPouwels et al., 1985, Cloning Vectors. A Laboratory Manual, Elsevier,New York) and the GATEWAY Vectors(lifetech.com/Content/Tech-Online/molecular_biology/manuals_pps/11797016.pdf;Life Technologies; Rockville, Md.). In the GATEWAY system the isolatednucleic acid of the invention, flanked by attB sequences, can berecombined through an integrase reaction with a GATEWAY vector such aspDONR201 containing attP sequences. This provides an entry vector forthe GATEWAY system containing the isolated nucleic acid of theinvention. This entry vector can be further recombined with othersuitably prepared expression control sequences, such as those of thepDC400 and pDC300 series described above. Many suitable expressioncontrol sequences are known in the art. General methods of expressingrecombinant polypeptides are also described in R. Kaufman, Methods inEnzymology 185, 537-566 (1990). As used herein “operably linked” meansthat the nucleic acid of the invention and an expression controlsequence are situated within a construct, vector, or cell in such a waythat the polypeptide encoded by the nucleic acid is expressed whenappropriate molecules (such as polymerases) are present. As oneembodiment of the invention, at least one expression control sequence isoperably linked to the nucleic acid of the invention in a recombinanthost cell or progeny thereof, the nucleic acid and/or expression controlsequence having been introduced into the host cell by transformation ortransfection, for example, or by any other suitable method. As anotherembodiment of the invention, at least one expression control sequence isintegrated into the genome of a recombinant host cell such that it isoperably linked to a nucleic acid sequence encoding a polypeptide of theinvention. In a further embodiment of the invention, at least oneexpression control sequence is operably linked to a nucleic acid of theinvention through the action of a trans-acting factor such as atranscription factor, either in vitro or in a recombinant host cell.

In addition, a sequence encoding an appropriate signal peptide (nativeor heterologous) can be incorporated into expression vectors. The choiceof signal peptide or leader can depend on factors such as the type ofhost cells in which the recombinant polypeptide is to be produced. Toillustrate, examples of heterologous signal peptides that are functionalin mammalian host cells include the signal sequence for interleukin-7(IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence forinterleukin-2 receptor described in Cosman et al., Nature 312:768(1984); the interleukin-4 receptor signal peptide described in EP367,566; the type I interleukin-1 receptor signal peptide described inU.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signalpeptide described in EP 460,846. A DNA sequence for a signal peptide(secretory leader) can be fused in frame to the nucleic acid sequence ofthe invention so that the DNA is initially transcribed, and the mRNAtranslated, into a fusion polypeptide comprising the signal peptide. Asignal peptide that is functional in the intended host cells is one thatpromotes insertion of the polypeptide into cell membranes, and mostpreferably, promotes extracellular secretion of the polypeptide fromthat host cell. The signal peptide is preferably cleaved from thepolypeptide upon membrane insertion or secretion of polypeptide from thecell. The skilled artisan will also recognize that the position(s) atwhich the signal peptide is cleaved can differ from that predicted bycomputer program, and can vary according to such factors as the type ofhost cells employed in expressing a recombinant polypeptide. Apolypeptide preparation can include a mixture of polypeptide moleculeshaving different N-terminal amino acids, resulting from cleavage of thesignal peptide at more than one site.

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69). Additional protocols using commercially available reagents, suchas Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine-Plus lipidreagent, can be used to transfect cells (Felgner et al., Proc. Natl.Acad. Sci. USA 84:7413-7417, 1987). In addition, electroporation can beused to transfect mammalian cells using conventional procedures, such asthose in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ed.Vol. 1-3, Cold Spring Harbor Laboratory Press, 1989). Selection ofstable transformants can be performed using methods known in the art,such as, for example, resistance to cytotoxic drugs. Kaufman et al.,Meth. in Enzymology 185:487-511, 1990, describes several selectionschemes, such as dihydrofolate reductase (DHFR) resistance. A suitablestrain for DHFR selection is CHO strain DX-B 11, which is deficient inDHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

Alternatively, IMX97018 gene products can be obtained via homologousrecombination, or “gene targeting,” techniques. Such techniques employthe introduction of exogenous transcription control elements (such asthe CMV promoter or the like) in a particular predetermined site on thegenome, to induce expression of the endogenous nucleic acid sequence ofinterest (see, for example, U.S. Pat. No. 5,272,071). The location ofintegration into a host chromosome or genome can be easily determined byone of skill in the art, given the known location and sequence of thegene. In a preferred embodiment, the present invention also contemplatesthe introduction of exogenous transcriptional control elements inconjunction with an amplifiable gene, to produce increased amounts ofthe gene product, again, without the need for isolation of the genesequence itself from the host cell.

A number of types of cells can act as suitable host cells for expressionof the polypeptide. Mammalian host cells include, for example, the COS-7line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (Rasmussen et al.,1998, Cytotechnology 28: 31), HeLa cells, BHK (ATCC CRL 10) cell lines,the CV1/EBNA cell line derived from the African green monkey kidney cellline CV1 (ATCC CCL 70) (McMahan et al., 1991, EMBO J. 10: 2821, 1991),human embryonic kidney cells such as 293, 293 EBNA or MSR 293, humanepidermal A431 cells, human Colo205 cells, other transformed primatecell lines, normal diploid cells, cell strains derived from in vitroculture of primary tissue, primary explants, HL-60, U937, HaK or Jurkatcells. Optionally, mammalian cell lines such as HepG2/3B, KB, NIH 3T3 orS49, for example, can be used for expression of the polypeptide when itis desirable to use the polypeptide in various signal transduction orreporter assays. Alternatively, it is possible to produce thepolypeptide in lower eukaryotes such as yeast or in prokaryotes such asbacteria. Suitable yeasts include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous polypeptides. Suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous polypeptides. If the polypeptide is made in yeast orbacteria, it may be desirable to modify the polypeptide producedtherein, for example by phosphorylation or glycosylation of theappropriate sites, in order to obtain the functional polypeptide. Suchcovalent attachments can be accomplished using known chemical orenzymatic methods. The polypeptide can also be produced by operablylinking the isolated nucleic acid of the invention to suitable controlsequences in one or more insect expression vectors, and employing aninsect expression system. Materials and methods for baculovirus/insectcell expression systems are commercially available in kit form from,e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Tex. Agricultural Experiment Station Bulletin No. 1555 (1987), andLuckow and Summers, Bio/Technology 6:47 (1988). Cell-free translationsystems could also be employed to produce polypeptides using RNAsderived from nucleic acid constructs disclosed herein. A host cell thatcomprises an isolated nucleic acid of the invention, preferably operablylinked to at least one expression control sequence, is a “recombinanthost cell”.

The polypeptide of the invention can be prepared by culturingtransformed host cells under culture conditions suitable to express therecombinant polypeptide. The resulting expressed polypeptide can then bepurified from such culture (i.e., from culture medium or cell extracts)using known purification processes, such as selective precipitation withvarious salts, gel filtration, and ion exchange chromatography. Thepurification of the polypeptide can also include an affinity columncontaining agents which will bind to the polypeptide; one or more columnsteps over such affinity resins as concanavalin A-agarose,heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more stepsinvolving hydrophobic interaction chromatography using such resins asphenyl ether, butyl ether, or propyl ether; or immunoaffinitychromatography using an antibody that specifically binds one or moreIMX97018 epitopes. Alternatively, the polypeptide of the invention canalso be expressed in a form which will facilitate purification. Forexample, it can be expressed as a fusion polypeptide, that is, it may befused with maltose binding polypeptide (MBP), glutathione-5-transferase(GST), thioredoxin (TRX), a polyHis peptide, and/or fragments thereof.Kits for expression and purification of such fusion polypeptides arecommercially available from New England BioLabs (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and InVitrogen, respectively. Thepolypeptide can also be tagged with an epitope and subsequently purifiedby using a specific antibody directed to such epitope. One such epitope(FLAG®) is commercially available from Kodak (New Haven, Conn.).Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the polypeptide. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous isolated recombinant polypeptide.The polypeptide thus purified is substantially free of other mammalianpolypeptides and is defined in accordance with the present invention asan “isolated polypeptide”; such isolated polypeptides of the inventioninclude isolated antibodies that bind to IMX97018 polypeptides,fragments, variants, binding partners etc. The polypeptide of theinvention can also be expressed as a product of transgenic animals,e.g., as a component of the milk of transgenic cows, goats, pigs, orsheep which are characterized by somatic or germ cells containing anucleotide sequence encoding the polypeptide.

It is also possible to utilize an affinity column comprising apolypeptide-binding polypeptide of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the invention. In this aspect of the invention,polypeptide-binding polypeptides, such as the anti-polypeptideantibodies of the invention or other polypeptides that can interact withthe polypeptide of the invention, can be bound to a solid phase supportsuch as a column chromatography matrix or a similar substrate suitablefor identifying, separating, or purifying cells that expresspolypeptides of the invention on their surface. Adherence ofpolypeptide-binding polypeptides of the invention to a solid phasecontacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with these polypeptide-bindingpolypeptides and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding polypeptides thereon. Cells having polypeptidesof the invention on their surface bind to the fixed polypeptide-bindingpolypeptide and unbound cells then are washed away. Thisaffinity-binding method is useful for purifying, screening, orseparating such polypeptide-expressing cells from solution. Methods ofreleasing positively selected cells from the solid phase are known inthe art and encompass, for example, the use of enzymes. Such enzymes arepreferably non-toxic and non-injurious to the cells and are preferablydirected to cleaving the cell-surface binding partner. Alternatively,mixtures of cells suspected of containing polypeptide-expressing cellsof the invention first can be incubated with a biotinylatedpolypeptide-binding polypeptide of the invention. The resulting mixturethen is passed through a column packed with avidin-coated beads, wherebythe high affinity of biotin for avidin provides the binding of thepolypeptide-binding cells to the beads. Use of avidin-coated beads isknown in the art. See Berenson, et al. J. Cell. Biochem., 10D:239(1986). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

The polypeptide can also be produced by known conventional chemicalsynthesis. Methods for constructing the polypeptides of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed polypeptide sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with IMX97018 polypeptides can possess biologicalproperties in common therewith, including IMX97018 polypeptide activity.Thus, they can be employed as biologically active or immunologicalsubstitutes for natural, purified polypeptides in screening oftherapeutic compounds and in immunological processes for the developmentof antibodies.

The desired degree of purity depends on the intended use of thepolypeptide. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no polypeptide bandscorresponding to other polypeptides are detectable upon analysis bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognizedby one skilled in the pertinent field that multiple bands correspondingto the polypeptide can be visualized by SDS-PAGE, due to differentialglycosylation, differential post-translational processing, and the like.Most preferably, the polypeptide of the invention is purified tosubstantial homogeneity, as indicated by a single polypeptide band uponanalysis by SDS-PAGE. The polypeptide band can be visualized by silverstaining, Coomassie blue staining, or (if the polypeptide isradiolabeled) by autoradiography.

Antagonists and Agonists of IMX97018 Polypeptides

Any method which neutralizes IMX97018 polypeptides or inhibitsexpression of the IMX97018 genes (either transcription or translation)can be used to reduce the biological activities of IMX97018polypeptides. In particular embodiments, antagonists inhibit the bindingof at least one IMX97018 polypeptide to cells, thereby inhibitingbiological activities induced by the binding of those IMX97018polypeptides to the cells. In certain other embodiments of theinvention, antagonists can be designed to reduce the level of endogenousIMX97018 gene expression, e.g., using well-known antisense or ribozymeapproaches to inhibit or prevent translation of IMX97018 mRNAtranscripts; triple helix approaches to inhibit transcription ofIMX97018 family genes; or targeted homologous recombination toinactivate or “knock out” the IMX97018 genes or their endogenouspromoters or enhancer elements. Such antisense, ribozyme, and triplehelix antagonists can be designed to reduce or inhibit eitherunimpaired, or if appropriate, mutant IMX97018 gene activity. Techniquesfor the production and use of such molecules are well known to those ofskill in the art.

Antisense RNA and DNA molecules act to directly block the translation ofmRNA by hybridizing to targeted mRNA and preventing polypeptidetranslation. Antisense approaches involve the design of oligonucleotides(either DNA or RNA) that are complementary to an IMX97018 mRNA. Theantisense oligonucleotides will bind to the complementary target genemRNA transcripts and prevent translation. Absolute complementarity,although preferred, is not required. A sequence “complementary” to aportion of a nucleic acid, as referred to herein, means a sequencehaving sufficient complementarity to be able to hybridize with thenucleic acid, forming a stable duplex (or triplex, as appropriate). Inthe case of double-stranded antisense nucleic acids, a single strand ofthe duplex DNA can thus be tested, or triplex formation can be assayed.The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid. Preferredoligonucleotides are complementary to the 5′ end of the message, e.g.,the 5′ untranslated sequence up to and including the AUG initiationcodon. However, oligonucleotides complementary to the 5′- or3′-non-translated, non-coding regions of the IMX97018 gene transcript,or to the coding regions, could be used in an antisense approach toinhibit translation of endogenous IMX97018 mRNA. Antisense nucleic acidsshould be at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixturesor derivatives or modified versions thereof, single-stranded ordouble-stranded. Chimeric oligonucleotides, oligonucleosides, or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment ofnucleotides is positioned between 5′ and 3′ “wing” segments of linkednucleosides and a second “open end” type wherein the “gap” segment islocated at either the 3′ or the 5′ terminus of the oligomeric compound(see, e.g., U.S. Pat. No. 5,985,664). Oligonucleotides of the first typeare also known in the art as “gapmers” or gapped oligonucleotides.Oligonucleotides of the second type are also known in the art as“hemimers” or “wingmers”. The oligonucleotide can be modified at thebase moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide can include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., 1989, Proc Natl Acad Sci U.S.A. 86: 6553-6556; Lemaitre et al.,1987, Proc Natl Acad Sci 84: 648-652; PCT Publication No. WO88/09810),or hybridization-triggered cleavage agents or intercalating agents.(See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). The antisense moleculesshould be delivered to cells which express the IMX97018 transcript invivo. A number of methods have been developed for delivering antisenseDNA or RNA to cells; e.g., antisense molecules can be injected directlyinto the tissue or cell derivation site, or modified antisensemolecules, designed to target the desired cells (e.g., antisense linkedto peptides or antibodies that specifically bind receptors or antigensexpressed on the target cell surface), can be administered systemically.However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation of endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous IMX97018 gene transcriptsand thereby prevent translation of the IMX97018 mRNA. For example, avector can be introduced in vivo such that it is taken up by a cell anddirects the transcription of an antisense RNA. Such a vector can remainepisomal or become chromosomally integrated, as long as it can betranscribed to produce the desired antisense RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Vectors can be plasmid, viral, or others known in the art, used forreplication and expression in mammalian cells.

Ribozyme molecules designed to catalytically cleave IMX97018 mRNAtranscripts can also be used to prevent translation of IMX97018 mRNA andexpression of IMX97018 polypeptides. (See, e.g., PCT InternationalPublication WO90/11364 and U.S. Pat. No. 5,824,519). The ribozymes thatcan be used in the present invention include hammerhead ribozymes(Haseloff and Gerlach, 1988, Nature, 334:585-591), RNA endoribonucleases(hereinafter “Cech-type ribozymes”) such as the one which occursnaturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA)and which has been extensively described by Thomas Cech andcollaborators (International Patent Application No. WO 88/04300; Beenand Cech, 1986, Cell, 47:207-216). As in the antisense approach, theribozymes can be composed of modified oligonucleotides (e.g. forimproved stability, targeting, etc.) and should be delivered to cellswhich express the IMX97018 polypeptide in vivo. A preferred method ofdelivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive pol III or pol II promoter, so thattransfected cells will produce sufficient quantities of the ribozyme todestroy endogenous IMX97018 messages and inhibit translation. Becauseribozymes, unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Alternatively, endogenous IMX97018 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the target gene (i.e., the target gene promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the target IMX97018 gene. (See generally, Helene, 1991, AnticancerDrug Des., 6(6), 569-584; Helene, et al., 1992, Ann. N.Y. Acad. Sci.,660, 27-36; and Maher, 1992, Bioassays 14(12), 807-815).

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of theinvention can be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Oligonucleotides can besynthesized by standard methods known in the art, e.g by use of anautomated DNA synthesizer (such as are commercially available fromBiosearch, Applied Biosystems, etc.). As examples, phosphorothioateoligonucleotides can be synthesized by the method of Stein et al., 1988,Nucl. Acids Res. 16:3209. Methylphosphonate oligonucleotides can beprepared by use of controlled pore glass polymer supports (Sarin et al.,1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451). Alternatively, RNAmolecules can be generated by in vitro and in vivo transcription of DNAsequences encoding the antisense RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Endogenous target gene expression can also be reduced by inactivating or“knocking out” the target gene or its promoter using targeted homologousrecombination (e.g., see Smithies, et al., 1985, Nature 317, 230-234;Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al., 1989,Cell 5, 313-321). For example, a mutant, non-functional target gene (ora completely unrelated DNA sequence) flanked by DNA homologous to theendogenous target gene (either the coding regions or regulatory regionsof the target gene) can be used, with or without a selectable markerand/or a negative selectable marker, to transfect cells that express thetarget gene in vivo. Insertion of the DNA construct, via targetedhomologous recombination, results in inactivation of the target gene.Such approaches are particularly suited in the agricultural field wheremodifications to ES (embryonic stem) cells can be used to generateanimal offspring with an inactive target gene (e.g., see Thomas andCapecchi, 1987 and Thompson, 1989, supra), or in model organisms such asCaenorhabditis elegans where the “RNA interference” (“RNAi”) technique(Grishok, Tabara, and Mello, 2000, Genetic requirements for inheritanceof RNAi in C. elegans, Science 287 (5462): 2494-2497), or theintroduction of transgenes (Demburg et al., 2000, Transgene-mediatedcosuppression in the C. elegans germ line, Genes Dev. 14 (13):1578-1583) are used to inhibit the expression of specific target genes.However this approach can be adapted for use in humans provided therecombinant DNA constructs are directly administered or targeted to therequired site in vivo using appropriate vectors such as viral vectors.

Organisms that have enhanced, reduced, or modified expression of thegene(s) corresponding to the nucleic acid sequences disclosed herein areprovided. The desired change in gene expression can be achieved throughthe use of antisense nucleic acids or ribozymes that bind and/or cleavethe mRNA transcribed from the gene (Albert and Morris, 1994, TrendsPharmacol. Sci. 15(7): 250-254; Lavarosky et al., 1997, Biochem. Mol.Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol.58: 1-39). Transgenic animals that have multiple copies of the gene(s)corresponding to the nucleic acid sequences disclosed herein, preferablyproduced by transformation of cells with genetic constructs that arestably maintained within the transformed cells and their progeny, areprovided. Transgenic animals that have modified genetic control regionsthat increase or reduce gene expression levels, or that change temporalor spatial patterns of gene expression, are also provided (see EuropeanPatent No. 0 649 464 B1). In addition, organisms are provided in whichthe gene(s) corresponding to the nucleic acid sequences disclosed hereinhave been partially or completely inactivated, through insertion ofextraneous sequences into the corresponding gene(s) or through deletionof all or part of the corresponding gene(s). Partial or complete geneinactivation can be accomplished through insertion, preferably followedby imprecise excision, of transposable elements (Plasterk, 1992,Bioessays 14(9): 629-633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA90(16): 7431-7435; Clark et al., 1994, Proc. Natl. Acad. Sci. USA 91(2):719-722), or through homologous recombination, preferably detected bypositive/negative genetic selection strategies (Mansour et al., 1988,Nature 336: 348-352; U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059;5,631,153; 5,614,396; 5,616,491; and 5,679,523). These organisms withaltered gene expression are preferably eukaryotes and more preferablyare mammals. Such organisms are useful for the development of non-humanmodels for the study of disorders involving the corresponding gene(s),and for the development of assay systems for the identification ofmolecules that interact with the polypeptide product(s) of thecorresponding gene(s).

Also encompassed within the invention are IMX97018 polypeptide variantswith partner binding sites that have been altered in conformation sothat (1) the IMX97018 variant will still bind to its partner(s), but aspecified small molecule will fit into the altered binding site andblock that interaction, or (2) the IMX97018 variant will no longer bindto its partner(s) unless a specified small molecule is present (see forexample Bishop et al., 2000, Nature 407: 395-401). Nucleic acidsencoding such altered IMX97018 polypeptides can be introduced intoorganisms according to methods described herein, and can replace theendogenous nucleic acid sequences encoding the corresponding IMX97018polypeptide. Such methods allow for the interaction of a particularIMX97018 polypeptide with its binding partners to be regulated byadministration of a small molecule compound to an organism, eithersystemically or in a localized manner.

The IMX97018 polypeptides themselves can also be employed in inhibitinga biological activity of IMX97018 in in vitro or in vivo procedures.Encompassed within the invention are AXH domains of IMX97018polypeptides that act as “dominant negative” inhibitors of nativeIMX97018 polypeptide function when expressed as fragments or ascomponents of fusion polypeptides. For example, a purified polypeptidedomain of the present invention can be used to inhibit binding ofIMX97018 polypeptides to endogenous binding partners. Such useeffectively would block IMX97018 polypeptide interactions and inhibitIMX97018 polypeptide activities. In still another aspect of theinvention, an antisense inhibitor is used to inhibit activation of theendogenous IMX97018 polypeptide.

In an alternative aspect, the invention further encompasses the use ofagonists of IMX97018 polypeptide activity to treat or ameliorate thesymptoms of a disease for which increased IMX97018 polypeptide activityis beneficial. Such diseases include but are not limited to neurologicaldisorders such as ataxias. In a preferred aspect, the invention entailsadministering compositions comprising an IMX97018 nucleic acid or anIMX97018 polypeptide to cells in vitro, to cells ex vivo, to cells invivo, and/or to a multicellular organism such as a vertebrate or mammal.Preferred therapeutic forms of IMX97018 are soluble forms, as describedabove. In still another aspect of the invention, the compositionscomprise administering an IMX97018-encoding nucleic acid for expressionof an IMX97018 polypeptide in a host organism for treatment of disease.Particularly preferred in this regard is expression in a human patientfor treatment of a dysfunction associated with aberrant (e.g.,decreased) endogenous activity of an IMX97018 family polypeptide.Furthermore, the invention encompasses the administration to cellsand/or organisms of compounds found to increase the endogenous activityof IMX97018 polypeptides. One example of compounds that increaseIMX97018 polypeptide activity are agonistic antibodies, preferablymonoclonal antibodies, that bind to IMX97018 polypeptides or bindingpartners, which may increase IMX97018 polypeptide activity by causingconstitutive intracellular signaling (or “ligand mimicking”), or bypreventing the binding of a native inhibitor of IMX97018 polypeptideactivity.

Antibodies to IMX97018 Polypeptides

Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). In the present invention, specifically bindingantibodies are those that will specifically recognize and bind withIMX97018 polypeptides, homologues, and variants, but not with othermolecules. In one preferred embodiment, the antibodies are specific forthe polypeptides of the present invention and do not cross-react withother polypeptides. In this manner, the IMX97018 polypeptides,fragments, variants, fusion polypeptides, etc., as set forth above canbe employed as “immunogens” in producing antibodies immunoreactivetherewith.

More specifically, the polypeptides, fragment, variants, fusionpolypeptides, etc. contain antigenic determinants or epitopes thatelicit the formation of antibodies. These antigenic determinants orepitopes can be either linear or conformational (discontinuous). Linearepitopes are composed of a single section of amino acids of thepolypeptide, while conformational or discontinuous epitopes are composedof amino acids sections from different regions of the polypeptide chainthat are brought into close proximity upon polypeptide folding (Janewayand Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed.1996)). Because folded polypeptides have complex surfaces, the number ofepitopes available is quite numerous; however, due to the conformationof the polypeptide and steric hindrances, the number of antibodies thatactually bind to the epitopes is less than the number of availableepitopes (Janeway and Travers, Immuno Biology 2:14 (Garland PublishingInc., 2nd ed. 1996)). Epitopes can be identified by any of the methodsknown in the art. Thus, one aspect of the present invention relates tothe antigenic epitopes of the polypeptides of the invention. Suchepitopes are useful for raising antibodies, in particular monoclonalantibodies, as described in more detail below. Additionally, epitopesfrom the polypeptides of the invention can be used as research reagents,in assays, and to purify specific binding antibodies from substancessuch as polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies can be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Kohler andMilstein, (U.S. Pat. No. 4,376,110); the human B-cell hybridomatechnique (Kozbor et al., 1984, J. Immunol. 133:3001-3005; Cole et al.,1983, Proc. Natl. Acad. Sci. USA 80:2026-2030); and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Hybridoma cell lines that producemonoclonal antibodies specific for the polypeptides of the invention arealso contemplated herein. Such hybridomas can be produced and identifiedby conventional techniques. The hybridoma producing the mAb of thisinvention can be cultivated in vitro or in vivo. Production of hightiters of mAbs in vivo makes this the presently preferred method ofproduction. One method for producing such a hybridoma cell linecomprises immunizing an animal with a polypeptide; harvesting spleencells from the immunized animal; fusing said spleen cells to a myelomacell line, thereby generating hybridoma cells; and identifying ahybridoma cell line that produces a monoclonal antibody that binds thepolypeptide. For the production of antibodies, various host animals canbe immunized by injection with one or more of the following: an IMX97018polypeptide, a fragment of an IMX97018 polypeptide, a functionalequivalent of an IMX97018 polypeptide, or a mutant form of an IMX97018polypeptide. Such host animals can include but are not limited torabbits, guinea pigs, mice, and rats. Various adjuvants can be used toincrease the immunologic response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjutants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. The monoclonal antibodies can be recovered by conventionaltechniques. Such monoclonal antibodies can be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

In addition, techniques developed for the production of “chimericantibodies” (Takeda et al., 1985, Nature, 314: 452-454; Morrison et al.,1984, Proc Natl Acad Sci USA 81: 6851-6855; Boulianne et al., 1984,Nature 312: 643-646; Neuberger et al., 1985, Nature 314: 268-270) bysplicing the genes from a mouse antibody molecule of appropriate antigenspecificity together with genes from a human antibody molecule ofappropriate biological activity can be used. A chimeric antibody is amolecule in which different portions are derived from different animalspecies, such as those having a variable region derived from a porcinemAb and a human immunoglobulin constant region. The monoclonalantibodies of the present invention also include humanized versions ofmurine monoclonal antibodies. Such humanized antibodies can be preparedby known techniques and offer the advantage of reduced immunogenicitywhen the antibodies are administered to humans. In one embodiment, ahumanized monoclonal antibody comprises the variable region of a murineantibody (or just the antigen binding site thereof) and a constantregion derived from a human antibody. Alternatively, a humanizedantibody fragment can comprise the antigen binding site of a murinemonoclonal antibody and a variable region fragment (lacking theantigen-binding site) derived from a human antibody. Procedures for theproduction of chimeric and further engineered monoclonal antibodiesinclude those described in Riechmann et al. (Nature 332:323, 1988), Liuet al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934,1989), and Winter and Harris (TIPS 14:139, Can, 1993). Useful techniquesfor humanizing antibodies are also discussed in U.S. Pat. No. 6,054,297.Procedures to generate antibodies transgenically can be found in GB2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806, and related patents.Preferably, for use in humans, the antibodies are human or humanized;techniques for creating such human or humanized antibodies are also wellknown and are commercially available from, for example, Medarex Inc.(Princeton, N.J.) and Abgenix Inc. (Fremont, Calif.). In anotherpreferred embodiment, fully human antibodies for use in humans areproduced by screening a library of human antibody variable domains usingeither phage display methods (Vaughan et al., 1998, Nat Biotechnol.16(6): 535-539; and U.S. Pat. No. 5,969,108), ribosome display methods(Schaffitzel et al., 1999, J Immunol Methods 231(1-2): 119-135), or mRNAdisplay methods (Wilson et al., 2001, Proc Natl Acad Sci USA 98(7):3750-3755).

Antigen-binding antibody fragments that recognize specific epitopes canbe generated by known techniques. For example, such fragments includebut are not limited to: the F(ab′)2 fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the (ab′)₂fragments. Alternatively, Fab expression libraries can be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778; Bird, 1988, Science 242:423-426; Huston et al.,1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989,Nature 334:544-546) can also be adapted to produce single chainantibodies against IMX97018 gene products. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide. Suchsingle chain antibodies can also be useful intracellularly (i.e., as‘intrabodies), for example as described by Marasco et al. (J. Immunol.Methods 231:223-238, 1999) for genetic therapy in HIV infection. Inaddition, antibodies to the IMX97018 polypeptide can, in turn, beutilized to generate anti-idiotype antibodies that “mimic” the IMX97018polypeptide and that may bind to the IMX97018 polypeptide's bindingpartners using techniques well known to those skilled in the art. (See,e.g., Greenspan & Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991,J. Immunol. 147(8):2429-2438).

Antibodies that are immunoreactive with the polypeptides of theinvention include bispecific antibodies (i.e., antibodies that areimmunoreactive with the polypeptides of the invention via a firstantigen binding domain, and also immunoreactive with a differentpolypeptide via a second antigen binding domain). A variety ofbispecific antibodies have been prepared, and found useful both in vitroand in vivo (see, for example, U.S. Pat. No. 5,807,706; and Cao andSuresh, 1998, Bioconjugate Chem 9: 635-644). Numerous methods ofpreparing bispecific antibodies are known in the art, including the useof hybrid-hybridomas such as quadromas, which are formed by fusing twodiffered hybridomas, and triomas, which are formed by fusing a hybridomawith a lymphocyte (Milstein and Cuello, 1983, Nature 305: 537-540; U.S.Pat. No. 4,474,893; and U.S. Pat. No. 6,106,833). U.S. Pat. No.6,060,285 discloses a process for the production of bispecificantibodies in which at least the genes for the light chain and thevariable portion of the heavy chain of an antibody having a firstspecificity are transfected into a hybridoma cell secreting an antibodyhaving a second specificity. Chemical coupling of antibody fragments hasalso been used to prepare antigen-binding molecules having specificityfor two different antigens (Brennan et al., 1985, Science 229: 81-83;Glennie et al., J Immunol., 1987, 139:2367-2375; and U.S. Pat. No.6,010,902). Bispecific antibodies can also be produced via recombinantmeans, for example, by using the leucine zipper moieties from the Fosand Jun proteins (which preferentially form heterodimers) as describedby Kostelny et al. (J. Immunol. 148:1547-4553; 1992). U.S. Pat. No.5,582,996 discloses the use of complementary interactive domains (suchas leucine zipper moieties or other lock and key interactive domainstructures) to facilitate heterodimer formation in the production ofbispecific antibodies. Tetravalent, bispecific molecules can be preparedby fusion of DNA encoding the heavy chain of an F(ab′)2 fragment of anantibody with either DNA encoding the heavy chain of a second F(ab′)2molecule (in which the CH1 domain is replaced by a CH3 domain), or withDNA encoding a single chain FV fragment of an antibody, as described inU.S. Pat. No. 5,959,083. Expression of the resultant fusion genes inmammalian cells, together with the genes for the corresponding lightchains, yields tetravalent bispecific molecules having specificity forselected antigens. Bispecific antibodies can also be produced asdescribed in U.S. Pat. No. 5,807,706. Generally, the method involvesintroducing a protuberance (constructed by replacing small amino acidside chains with larger side chains) at the interface of a firstpolypeptide and a corresponding cavity (prepared by replacing largeamino acid side chains with smaller ones) in the interface of a secondpolypeptide. Moreover, single-chain variable fragments (sFvs) have beenprepared by covalently joining two variable domains; the resultingantibody fragments can form dimers or trimers, depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Protein Engineering 10:423-433).

Screening procedures by which such antibodies can be identified are wellknown, and can involve immunoaffinity chromatography, for example.Antibodies can be screened for agonistic (i.e., ligand-mimicking)properties. Such antibodies, upon binding to cell surface IMX97018,induce biological effects (e.g., transduction of biological signals)similar to the biological effects induced when the IMX97018 bindingpartner binds to cell surface IMX97018. Agonistic antibodies can be usedto induce IMX97018-mediated cell stimulatory pathways or intercellularcommunication. Bispecific antibodies can be identified by screening withtwo separate assays, or with an assay wherein the bispecific antibodyserves as a bridge between the first antigen and the second antigen (thelatter is coupled to a detectable moiety). Bispecific antibodies thatbind IMX97018 polypeptides of the invention via a first antigen bindingdomain will be useful in diagnostic applications. Examples ofpolypeptides (or other antigens) that the inventive bispecificantibodies bind via a second antigen binding domain include LANP, otherleucine-rich polypeptides, and polyglutamine-containing polypeptides.

Also provided herein are conjugates comprising a detectable (e.g.,diagnostic) or therapeutic agent, attached to the antibody. Examples ofsuch agents are presented above. The conjugates find use in in vitro orin vivo procedures. The antibodies of the invention can also be used inassays to detect the presence of the polypeptides or fragments of theinvention, either in vitro or in vivo. The antibodies also can beemployed in purifying polypeptides or fragments of the invention byimmunoaffinity chromatography.

Rational Design of Compounds that Interact with IMX97018 Polypeptides

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact, e.g., inhibitors, agonists, antagonists, etc. Anyof these examples can be used to fashion drugs which are more active orstable forms of the polypeptide or which enhance or interfere with thefunction of a polypeptide in vivo (Hodgson J (1991) Biotechnology9:19-21). In one approach, the three-dimensional structure of apolypeptide of interest, or of a polypeptide-inhibitor complex, isdetermined by x-ray crystallography, by nuclear magnetic resonance, orby computer homology modeling or, most typically, by a combination ofthese approaches. Both the shape and charges of the polypeptide must beascertained to elucidate the structure and to determine active site(s)of the molecule. Less often, useful information regarding the structureof a polypeptide may be gained by modeling based on the structure ofhomologous polypeptides. In both cases, relevant structural informationis used to design analogous IMX97018-like molecules, to identifyefficient inhibitors, or to identify small molecules that bind IMX97018polypeptides. Useful examples of rational drug design include moleculeswhich have improved activity or stability as shown by Braxton S andWells J A (1992 Biochemistry 31:7796-7801) or which act as inhibitors,agonists, or antagonists of native peptides as shown by Athauda S B etal (1993 J Biochem 113:742-746). The use of IMX97018 polypeptidestructural information in molecular modeling software systems to assistin inhibitor design and in studying inhibitor-IMX97018 polypeptideinteraction is also encompassed by the invention. A particular method ofthe invention comprises analyzing the three dimensional structure ofIMX97018 polypeptides for likely binding sites of substrates,synthesizing a new molecule that incorporates a predictive reactivesite, and assaying the new molecule as described further herein.

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described further herein, and then to solve itscrystal structure. This approach, in principle, yields a pharmacore uponwhich subsequent drug design can be based. It is possible to bypasspolypeptide crystallography altogether by generating anti-idiotypicantibodies (anti-ids) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-ids would be expected to be an analog of the original antigen. Theanti-id could then be used to identify and isolate peptides from banksof chemically or biologically produced peptides. The isolated peptideswould then act as the pharmacore.

Assays of IMX97018 Polypeptide Activities

The purified IMX97018 polypeptides of the invention (includingpolypeptides, polypeptides, fragments, variants, oligomers, and otherforms) are useful in a variety of assays. For example, the IMX97018molecules of the present invention can be used to identify bindingpartners of IMX97018 polypeptides, which can also be used to modulateintercellular communication, cell stimulation, or immune cell activity.Alternatively, they can be used to identify non-binding-partnermolecules or substances that modulate intercellular communication, cellstimulatory pathways, or immune cell activity.

Assays to Identify Binding Partners. Polypeptides of the IMX97018 familyand fragments thereof can be used to identify binding partners. Forexample, they can be tested for the ability to bind a candidate bindingpartner in any suitable assay, such as a conventional binding assay. Toillustrate, the IMX97018 polypeptide can be labeled with a detectablereagent (e.g., a radionuclide, chromophore, enzyme that catalyzes acolorimetric or fluorometric reaction, and the like). The labeledpolypeptide is contacted with cells expressing the candidate bindingpartner. The cells then are washed to remove unbound labeledpolypeptide, and the presence of cell-bound label is determined by asuitable technique, chosen according to the nature of the label.

One example of a binding assay procedure is as follows. A recombinantexpression vector containing the candidate binding partner cDNA isconstructed; the candidate binding partner can be part of a fusionprotein construct that includes a leader peptide and/or a transmembranedomain, so that the candidate binding partner when expressed is locatedon the exterior of the cell surface. CV1-EBNA-1 cells in 10 cm² dishesare transfected with this recombinant expression vector. CV-1/EBNA-1cells (ATCC CRL 10478) constitutively express EBV nuclear antigen-1driven from the CMV Immediate-early enhancer/promoter. CV1-EBNA-1 wasderived from the African Green Monkey kidney cell line CV-1 (ATCC CCL70), as described by McMahan et al., (EMBO J. 10:2821, 1991). Thetransfected cells are cultured for 24 hours, and the cells in each dishthen are split into a 24-well plate. After culturing an additional 48hours, the transfected cells (about 4×10⁴ cells/well) are washed withBM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovineserum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50mg/ml nonfat dry milk has been added. The cells then are incubated for 1hour at 37° C. with various concentrations of, for example, a solublepolypeptide/Fc fusion polypeptide made as set forth above. Cells thenare washed and incubated with a constant saturating concentration of a¹²⁵I-mouse anti-human IgG in binding medium, with gentle agitation for 1hour at 37° C. After extensive washing, cells are released viatrypsinization. The mouse anti-human IgG employed above is directedagainst the Fc region of human IgG and can be obtained from JacksonImmunoresearch Laboratories, Inc., West Grove, Pa. The antibody isradioiodinated using the standard chloramine-T method. The antibody willbind to the Fc portion of any polypeptide/Fc polypeptide that has boundto the cells. In all assays, non-specific binding of ¹²⁵I-antibody isassayed in the absence of the Fc fusion polypeptide/Fc, as well as inthe presence of the Fc fusion polypeptide and a 200-fold molar excess ofunlabeled mouse anti-human IgG antibody. Cell-bound ¹²⁵I-antibody isquantified on a Packard Autogamma counter. Affinity calculations(Scatchard, Ann. N Y Acad. Sci. 51:660, 1949) are generated on RS/1 (BBNSoftware, Boston, Mass.) run on a Microvax computer. Binding can also bedetected using methods that are well suited for high-throughputscreening procedures, such as scintillation proximity assays (Udenfriendet al., 1985, Proc Natl Acad Sci USA 82: 8672-8676), homogeneoustime-resolved fluorescence methods (Park et al., 1999, Anal Biochem 269:94-104), fluorescence resonance energy transfer (FRET) methods (Clegg RM, 1995, Curr Opin Biotechnol 6: 103-110), or methods that measure anychanges in surface plasmon resonance when a bound polypeptide is exposedto a potential binding partner, using for example a biosensor such asthat supplied by Biacore AB (Uppsala, Sweden). Compounds that can beassayed for binding to IMX97018 polypeptides include but are not limitedto small organic molecules, such as those that are commerciallyavailable—often as part of large combinatorial chemistry compound‘libraries’—from companies such as Sigma-Aldrich (St. Louis, Mo.),Arqule (Woburn, Mass.), Enzymed (Iowa City, Iowa), Maybridge ChemicalCo. (Trevillett, Cornwall, UK), MDS Panlabs (Bothell, Wash.),Pharmacopeia (Princeton, N.J.), and Trega (San Diego, Calif.). Preferredsmall organic molecules for screening using these assays are usuallyless than 10K molecular weight and can possess a number ofphysicochemical and pharmacological properties which enhance cellpenetration, resist degradation, and/or prolong their physiologicalhalf-lives (Gibbs, J., 1994, Pharmaceutical Research in MolecularOncology, Cell 79(2): 193-198). Compounds including natural products,inorganic chemicals, and biologically active materials such as proteinsand toxins can also be assayed using these methods for the ability tobind to IMX97018 polypeptides.

Yeast Two-Hybrid or “Interaction Trap” Assays. Where the IMX97018polypeptide binds or potentially binds to another polypeptide (such as,for example, in a receptor-ligand interaction), the nucleic acidencoding the IMX97018 polypeptide can also be used in interaction trapassays (such as, for example, that described in Gyuris et al., Cell75:791-803 (1993)) to identify nucleic acids encoding the otherpolypeptide with which binding occurs or to identify inhibitors of thebinding interaction. Polypeptides involved in these binding interactionscan also be used to screen for peptide or small molecule inhibitors oragonists of the binding interaction.

Competitive Binding Assays. Another type of suitable binding assay is acompetitive binding assay. To illustrate, biological activity of avariant can be determined by assaying for the variant's ability tocompete with the native polypeptide for binding to the candidate bindingpartner. Competitive binding assays can be performed by conventionalmethodology. Reagents that can be employed in competitive binding assaysinclude radiolabeled IMX97018 and intact cells expressing IMX97018(endogenous or recombinant) on the cell surface. For example, aradiolabeled soluble IMX97018 fragment can be used to compete with asoluble IMX97018 variant for binding to cell surface receptors. Insteadof intact cells, one could substitute a soluble binding partner/Fcfusion polypeptide bound to a solid phase through the interaction ofPolypeptide A or Polypeptide G (on the solid phase) with the Fc moiety.Chromatography columns that contain Polypeptide A and Polypeptide Ginclude those available from Pharmacia Biotech, Inc., Piscataway, N.J.

Diagnostic and Other Uses of IMX97018 Polypeptides and Nucleic Acids

The nucleic acids encoding the IMX97018 polypeptides provided by thepresent invention can be used for numerous diagnostic or other usefulpurposes. The nucleic acids of the invention can be used to expressrecombinant polypeptide for analysis, characterization or therapeuticuse; as markers for tissues in which the corresponding polypeptide ispreferentially expressed (either constitutively or at a particular stageof tissue differentiation or development or in disease states); asmolecular weight markers on Southern gels; as chromosome markers or tags(when labeled) to identify chromosome 16q22.3 or to map related genepositions; to compare with endogenous DNA sequences in patients toidentify potential polyglutamine-related genetic disorders, such asspinocerebellar ataxias; as probes to hybridize and thus discover novel,related DNA sequences; as a source of information to derive PCR primersfor genetic fingerprinting; as a probe to “subtract-out” known sequencesin the process of discovering other novel nucleic acids; for selectingand making oligomers for attachment to a “gene chip” or other support,including for examination of expression patterns; to raiseanti-polypeptide antibodies using DNA immunization techniques; as anantigen to raise anti-DNA antibodies or elicit another immune response,and for gene therapy. Uses of IMX97018 polypeptides and fragmentedpolypeptides include, but are not limited to, the following: purifyingpolypeptides and measuring the activity thereof; delivery agents;therapeutic and research reagents; molecular weight and isoelectricfocusing markers; controls for peptide fragmentation; identification ofunknown polypeptides; and preparation of antibodies. Any or all nucleicacids suitable for these uses are capable of being developed intoreagent grade or kit format for commercialization as products. Methodsfor performing the uses listed above are well known to those skilled inthe art. References disclosing such methods include without limitation“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987

Probes and Primers. Among the uses of the disclosed IMX97018 nucleicacids, and combinations of fragments thereof, is the use of fragments asprobes or primers. Such fragments generally comprise at least about 17contiguous nucleotides of a DNA sequence. In other embodiments, a DNAfragment comprises at least 30, or at least 60, contiguous nucleotidesof a DNA sequence. The basic parameters affecting the choice ofhybridization conditions and guidance for devising suitable conditionsare set forth by Sambrook et al., 1989 and are described in detailabove. Using knowledge of the genetic code in combination with the aminoacid sequences set forth above, sets of degenerate oligonucleotides canbe prepared. Such oligonucleotides are useful as primers, e.g., inpolymerase chain reactions (PCR), whereby DNA fragments are isolated andamplified. In certain embodiments, degenerate primers can be used asprobes for non-human genetic libraries. Such libraries would include butare not limited to cDNA libraries, genomic libraries, and evenelectronic EST (express sequence tag) or DNA libraries. Homologoussequences identified by this method would then be used as probes toidentify non-human IMX97018 homologues.

Chromosome Mapping. The nucleic acids encoding IMX97018 polypeptides,and the disclosed fragments and combinations of these nucleic acids, canbe used by those skilled in the art using well-known techniques toidentify human chromosome 16, and in particular the 16q22.3 region ofthat chromosome, to which these nucleic acids map. Useful techniquesinclude, but are not limited to, using the sequence or portions,including oligonucleotides, as a probe in various well-known techniquessuch as radiation hybrid mapping (high resolution), in situhybridization to chromosome spreads (moderate resolution), and Southernblot hybridization to hybrid cell lines containing individual humanchromosomes (low resolution).

Diagnostics and Gene Therapy. The nucleic acids encoding IMX97018polypeptides, and the disclosed fragments and combinations of thesenucleic acids can be used by one skilled in the art as a positionalmarker to map other genes of unknown location using well-knowntechniques. The nucleic acid encoding IMX97018 polypeptide has beenlocated to a particular position on human chromosome 16q22.3 and can beused to map genetic disorders relative to that location, or to moreprecisely map human genetic disorders already known to be linked tochromosome 16. For example, the following genetic disorders are locatednear the chromosomal position of IX97018-encoding sequences, but thegenes associated with these disorders have not yet been identified:Zonular Cataract (also called Perinuclear Cataract, Lamellar Cataract,Marner Cataract); Wilms Tumor Gene 3 (WT3); Acute Myelogenous Leukemia(AMLCR2), North American Indian Childhood Cirrhosis (CIRHIA, NAIC). Asdiscussed below, another genetic disorder that maps closely toIMX97018-encoding sequences is Spinocerebellar Ataxia 4 (SCA4).IMX97018-encoding nucleic acids can be used to analyze geneticabnormalities associated with the SCA4 disorder, for example, enablingone of skill in the art to distinguish SCA4 kindreds in whichchromosomal regions comprising IMX97018-encoding sequences arerearranged or deleted. There is substantial utility in nucleic acidsthat can be used to confirm or to eliminate SCA4 as a genetic factor fora kindred presenting with hereditary ataxia. For example, elimination ofthe known forms of SCA is the first step in diagnosing a family with ahereditary ataxia (see, for example, Devos et al., 2001, Neurology 56:234-238). Further, assuming that the SCA4 gene overlaps with orcorresponds to UMX97018-encoding sequences, the IMX97018-encodingnucleic acids can be used to identify genetic alterations in the SCA4gene at the nucleotide level. Additionally, the IMX97018-encodingnucleic acids can be used in developing treatments for any disordermediated (directly or indirectly) by defective, or insufficient amountsof, the genes corresponding to the nucleic acids of the invention.Disclosure herein of native nucleotide sequences permits the detectionof defective genes, and the replacement thereof with normal genes.Defective genes can be detected in in vitro diagnostic assays, and bycomparison of a native nucleotide sequence disclosed herein with that ofa gene derived from a person suspected of harboring a defect in thisgene.

Methods of Screening for Binding Partners. The IMX97018 polypeptides ofthe invention each can be used as reagents in methods to screen for oridentify binding partners. For example, the IMX97018 polypeptides can beattached to a solid support material and may bind to their bindingpartners in a manner similar to affinity chromatography. In particularembodiments, a polypeptide is attached to a solid support byconventional procedures. As one example, chromatography columnscontaining functional groups that will react with functional groups onamino acid side chains of polypeptides are available (Pharmacia Biotech,Inc., Piscataway, N.J.). In an alternative, a polypeptide/Fc polypeptide(as discussed above) is attached to protein A- or protein G-containingchromatography columns through interaction with the Fc moiety. PurifiedIMX97018 polypeptides are bound to a solid phase such as a columnchromatography matrix or a similar suitable substrate. For example,magnetic microspheres can be coated with the polypeptides and held in anincubation vessel through a magnetic field. Suspensions of cell mixturescontaining potential binding-partner-expressing cells are contacted withthe solid phase having the polypeptides thereon. Cells expressing thebinding partner on the cell surface bind to the fixed polypeptides, andunbound cells are washed away. Alternatively, IMX97018 polypeptides canbe conjugated to a detectable moiety, then incubated with cells to betested for binding partner expression. After incubation, unbound labeledmatter is removed and the presence or absence of the detectable moietyon the cells is determined. In a further alternative, mixtures of cellssuspected of expressing the binding partner are incubated withbiotinylated polypeptides. Incubation periods are typically at least onehour in duration to ensure sufficient binding. The resulting mixturethen is passed through a column packed with avidin-coated beads, wherebythe high affinity of biotin for avidin provides binding of the desiredcells to the beads. Procedures for using avidin-coated beads are known(see Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Washing toremove unbound material, and the release of the bound cells, areperformed using conventional methods. In some instances, the abovemethods for screening for or identifying binding partners may also beused or modified to isolate or purify such binding partner molecules orcells expressing them.

Measuring Biological Activity. Polypeptides also find use in measuringthe biological activity of IMX97018-binding polypeptides in terms oftheir binding affinity. The polypeptides thus can be employed by thoseconducting “quality assurance” studies, e.g., to monitor shelf life andstability of polypeptide under different conditions. For example, thepolypeptides can be employed in a binding affinity study to measure thebiological activity of a binding partner polypeptide that has beenstored at different temperatures, or produced in different cell types.The polypeptides also can be used to determine whether biologicalactivity is retained after modification of a binding partner polypeptide(e.g., chemical modification, truncation, mutation, etc.). The bindingaffinity of the modified polypeptide is compared to that of anunmodified binding polypeptide to detect any adverse impact of themodifications on biological activity of the binding polypeptide. Thebiological activity of a binding polypeptide thus can be ascertainedbefore it is used in a research study, for example.

Carriers and Delivery Agents. The polypeptides also find use as carriersfor delivering agents attached thereto to cells comprising identifiedbinding partners. In one method of the invention, the IMX97018polypeptide is covalently linked to a protein-transduction domain (PTD)such as, but not limited to, TAT, Antp, or VP22 (Schwarze et al., 2000,Cell Biology 10: 290-295). The polypeptides thus can be used to deliverdiagnostic or therapeutic agents into such cells in in vitro or in vivoprocedures. Detectable (diagnostic) and therapeutic agents that can beattached to a polypeptide include, but are not limited to, toxins, othercytotoxic agents, drugs, radionuclides, chromophores, enzymes thatcatalyze a colorimetric or fluorometric reaction, and the like, with theparticular agent being chosen according to the intended application.Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonasaeruginosa exotoxin A, ribosomal inactivating polypeptides, mycotoxinssuch as trichothecenes, and derivatives and fragments (e.g., singlechains) thereof. Radionuclides suitable for diagnostic use include, butare not limited to, ¹²³I, ¹³¹I, ^(99m)Tc, ¹¹¹In, and ⁷⁶Br. Examples ofradionuclides suitable for therapeutic use are ¹³¹I, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re,¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu. Such agents can be attachedto the polypeptide by any suitable conventional procedure. Thepolypeptide comprises functional groups on amino acid side chains thatcan be reacted with functional groups on a desired agent to formcovalent bonds, for example. Alternatively, the polypeptide or agent canbe derivatized to generate or attach a desired reactive functionalgroup. The derivatization can involve attachment of one of thebifunctional coupling reagents available for attaching various moleculesto polypeptides (Pierce Chemical Company, Rockford, Ill.). A number oftechniques for radiolabeling polypeptides are known. Radionuclide metalscan be attached to polypeptides by using a suitable bifunctionalchelating agent, for example. Conjugates comprising polypeptides and asuitable diagnostic or therapeutic agent (preferably covalently linked)are thus prepared. The conjugates are administered or otherwise employedin an amount appropriate for the particular application.

Treating Diseases with IMX97018 Polypeptides and Antagonists Thereof

The IMX97018 polypeptides, fragments, variants, antagonists, agonists,antibodies, and binding partners of the invention are likely to beuseful for treating medical conditions and diseases including, but notlimited to, neurological and SCA conditions as described further herein.The therapeutic molecule or molecules to be used will depend on theetiology of the condition to be treated and the biological pathwaysinvolved, and variants, fragments, and binding partners of IMX97018polypeptides may have effects similar to or different from IMX97018polypeptides. For example, an antagonist of the SCA-promoting activityof IMX97018 polypeptides containing polyglutamine tracts (‘IMX97018polyQpolypeptides’) can be selected for treatment of conditions involvingexpansion of polyglutamine tracts, but a particular fragment of a givenIMX97018 polypeptide itself, such as a fragment comprising the AXHdomain, may also act as an effective dominant negative antagonist ofthat activity. Therefore, in the following paragraphs “IMX97018polypeptides or antagonists” refers to all IMX97018 polypeptides,fragments, variants, antagonists, agonists, antibodies, and bindingpartners etc. of the invention, and it is understood that a specificmolecule or molecules can be selected from those provided as embodimentsof the invention by individuals of skill in the art, according to thebiological and therapeutic considerations described herein.

Antagonists of IMX97018polyQ polypeptides and IMX97018 polypeptides andagonists are useful for treating SCA-related disorders includingSpinocerebellar atrophy I (SCA1), Olivopontocerebellar atrophy I(OPCA1), Menzel type OPCA, Spinocerebellar atrophy IV (SCA4), andSpinocerebellar ataxia, autosomal dominant, with sensory axonalneuropathy; ataxia; progressive loss of muscle coordination and tone;dementia; Alzheimer's disease, disorders of vertebral disk or columndevelopment; Bell's palsy; transmissible dementia, includingCreutzfeld-Jacob disease; demyelinating neuropathy; Guillain-Barresyndrome; myasthenia gravis; silent cerebral ischemia; sleep disorders,including insomnia, narcolepsy, and sleep apnea; chronic neuronaldegeneration; stroke, including cerebral ischemic diseases; andneurological conditions such as those associated with the followingsymptoms: progressive cerebellar ataxia, cerebellar ataxia of gait andlimbs, supranuclear ophthalmoplegia, pyramidal or extrapyramidal signs,mild dementia, peripheral neuropathy, macular and retinal degeneration,upper motor neuron signs, extensor plantar responses, involuntarychoreiform movements, abnormal eye movements, abnormal saccade amplitudeor velocity, presence of gaze-evoked nystagmus, hypermetria, lowerbulbar palsies, hyperreflexia, scanning and explosive speech,incoordination, slow motor-nerve conduction, atrophy of the cerebellum,pons and olives, degeneration of lower cranial nerve nuclei, and atrophyof the dorsal columns and spinocerebellar tracts, abnormal deep tendonreflexes, reduced aspartic acid and/or markedly elevated taurine contentin brain tissue, reduction in platelet glutamate dehydrogenase activity,lack of activation of GDH by ADP in either the presence or the absenceof Triton, neuronal loss from the pars compacta of the substantia nigraor in the locus coeruleus, severe atrophy of the dentatorubral pathways,severe loss of Purkinje cells and degeneration of the olivocerebellarpathways, atrophy of the nucleus pontis, marked atrophy of Clarkecolumns and the spinocerebellar tracts, diplopia, severe spasticity orpronounced peripheral neuropathy, impaired temperature discrimination,abnormal peripheral and central motor conduction times in motor evokedpotentials, pontine and cerebellar atrophy, enlargement of the fourthventricle, gait disturbance, difficulty with fine motor tasks,dysarthria, vibratory and joint position sense loss, pinprick-sensationloss, loss of ankle-jerk reflexes, loss of knee-jerk reflexes, andcomplete areflexia. In addition, provided herein is the use ofantagonists of IMX97018polyQ polypeptides and IMX97018 polypeptides andagonists to treat AIDS-related neurological conditions, such as AIDSdementia complex. Antagonists of IMX97018polyQ polypeptides are alsouseful for treating polyglutamine-related disorders including fragile Xsyndrome, myotonic dystrophy, Kennedy spinal and bulbar muscularatrophy, and Huntington disease.

Antagonists of IMX97018 polypeptides and IMX97018polyQ polypeptides andagonists are useful for promoting cell death, and particularly forpromoting neural cell death via a non-apoptotic mechanism. Providedherein are methods for using antagonists of IMX97018 polypeptides,IMX97018polyQ polypeptides, IMX97018polyQ polypeptide agonists,compositions or combination therapies to treat various neural oncologicdisorders including brain tumors, glioma, glioblastoma, astrocytoma,oligodendroglioma, ependymoma, ganglioglioma, medulloblastoma,neuroectodermal tumors, and pilocytic astrocytoma.

Administration of IMX97018 Polypeptides and Antagonists Thereof

This invention provides compounds, compositions, and methods fortreating a patient, preferably a mammalian patient, and most preferablya human patient, who is suffering from a medical disorder, and inparticular an IM4×97018-mediated disorder. Such IMX97018-mediateddisorders include conditions caused (directly or indirectly) orexacerbated by binding between IMX97018 and a binding partner. Forpurposes of this disclosure, the terms “illness,” “disease,” “medicalcondition,” “abnormal condition” and the like are used interchangeablywith the term “medical disorder.” The terms “treat”, “treating”, and“treatment” used herein includes curative, preventative (e.g.,prophylactic) and palliative or ameliorative treatment. For suchtherapeutic uses, IMX97018 polypeptides and fragments, IMX97018 nucleicacids encoding the IMX97018 family polypeptides, and/or agonists orantagonists of the IMX97018 polypeptide such as antibodies can beadministered to the patient in need through well-known means.Compositions of the present invention can contain a polypeptide in anyform described herein, such as native polypeptides, variants,derivatives, oligomers, and biologically active fragments. In particularembodiments, the composition comprises a soluble polypeptide or anoligomer comprising soluble IMX97018 polypeptides.

Therapeutically Effective Amount. In practicing the method of treatmentor use of the present invention, a therapeutically effective amount of atherapeutic agent of the present invention is administered to a patienthaving a condition to be treated, preferably to treat or amelioratediseases associated with the activity of an IMX97018 family polypeptide.“Therapeutic agent” includes without limitation any of the EMX97018polypeptides, fragments, and variants; nucleic acids encoding theIMX97018 family polypeptides, fragments, and variants; agonists orantagonists of the IMX97018 polypeptides such as antibodies; IMX97018polypeptide binding partners; complexes formed from the IMX97018 familypolypeptides, fragments, variants, and binding partners, etc. As usedherein, the term “therapeutically effective amount” means the totalamount of each therapeutic agent or other active component of thepharmaceutical composition or method that is sufficient to show ameaningful patient benefit, i.e., treatment, healing, prevention oramelioration of the relevant medical condition, or an increase in rateof treatment, healing, prevention or amelioration of such conditions.When applied to an individual therapeutic agent or active ingredient,administered alone, the term refers to that ingredient alone. Whenapplied to a combination, the term refers to combined amounts of theingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously. As used herein, the phrase“administering a therapeutically effective amount” of a therapeuticagent means that the patient is treated with said therapeutic agent inan amount and for a time sufficient to induce an improvement, andpreferably a sustained improvement, in at least one indicator thatreflects the severity of the disorder. An improvement is considered“sustained” if the patient exhibits the improvement on at least twooccasions separated by one or more days, or more preferably, by one ormore weeks. The degree of improvement is determined based on signs orsymptoms, and determinations can also employ questionnaires that areadministered to the patient, such as quality-of-life questionnaires.Various indicators that reflect the extent of the patient's illness canbe assessed for determining whether the amount and time of the treatmentis sufficient. The baseline value for the chosen indicator or indicatorsis established by examination of the patient prior to administration ofthe first dose of the therapeutic agent. Preferably, the baselineexamination is done within about 60 days of administering the firstdose. If the therapeutic agent is being administered to treat acutesymptoms, the first dose is administered as soon as practically possibleafter the injury has occurred. Improvement is induced by administeringtherapeutic agents such as IMX97018 polypeptides or antagonists untilthe patient manifests an improvement over baseline for the chosenindicator or indicators. In treating chronic conditions, this degree ofimprovement is obtained by repeatedly administering this medicament overa period of at least a month or more, e.g., for one, two, or threemonths or longer, or indefinitely. A period of one to six weeks, or evena single dose, often is sufficient for treating injuries or other acuteconditions. Although the extent of the patient's illness after treatmentmay appear improved according to one or more indicators, treatment maybe continued indefinitely at the same level or at a reduced dose orfrequency. Once treatment has been reduced or discontinued, it later maybe resumed at the original level if symptoms should reappear.

Dosing. One skilled in the pertinent art will recognize that suitabledosages will vary, depending upon such factors as the nature andseverity of the disorder to be treated, the patient's body weight, age,general condition, and prior illnesses and/or treatments, and the routeof administration. Preliminary doses can be determined according toanimal tests, and the scaling of dosages for human administration isperformed according to art-accepted practices such as standard dosingtrials. For example, the therapeutically effective dose can be estimatedinitially from cell culture assays. The dosage will depend on thespecific activity of the compound and can be readily determined byroutine experimentation. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture,while minimizing toxicities. Such information can be used to moreaccurately determine useful doses in humans. Ultimately, the attendingphysician will decide the amount of polypeptide of the present inventionwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of polypeptide of the presentinvention and observe, the patient's response. Larger doses ofpolypeptide of the present invention can be administered until theoptimal therapeutic effect is obtained for the patient, and at thatpoint the dosage is not increased further. It is contemplated that thevarious pharmaceutical compositions used to practice the method of thepresent invention should contain about 0.01 ng to about 100 mg(preferably about 0.1 ng to about 10 mg, more preferably about 0.1microgram to about 1 mg) of polypeptide of the present invention per kgbody weight. In one embodiment of the invention, IMX97018 polypeptidesor antagonists are administered one time per week to treat the variousmedical disorders disclosed herein, in another embodiment isadministered at least two times per week, and in another embodiment isadministered at least three times per week. If injected, the effectiveamount of IMX97018 polypeptides or antagonists per adult dose rangesfrom 1-20 mg/m², and preferably is about 5-12 mg/m². Alternatively, aflat dose can be administered, whose amount may range from 5-100mg/dose. Exemplary dose ranges for a flat dose to be administered bysubcutaneous injection are 5-25 mg/dose, 25-50 mg/dose and 50-100mg/dose. In one embodiment of the invention, the various indicationsdescribed below are treated by administering a preparation acceptablefor injection containing IMX97018 polypeptides or antagonists at 25mg/dose, or alternatively, containing 50 mg per dose. The 25 mg or 50 mgdose can be administered repeatedly, particularly for chronicconditions. If a route of administration other than injection is used,the dose is appropriately adjusted in accord with standard medicalpractices. In many instances, an improvement in a patient's conditionwill be obtained by injecting a dose of about 25 mg of IMX97018polypeptides or antagonists one to three times per week over a period ofat least three weeks, or a dose of 50 mg of IMX97018 polypeptides orantagonists one or two times per week for at least three weeks, thoughtreatment for longer periods may be necessary to induce the desireddegree of improvement. For incurable chronic conditions, the regimen canbe continued indefinitely, with adjustments being made to dose andfrequency if such are deemed necessary by the patient's physician. Theforegoing doses are examples for an adult patient who is a person who is18 years of age or older. For pediatric patients (age 4-17), a suitableregimen involves the subcutaneous injection of 0.4 mg/kg, up to amaximum dose of 25 mg of IMX97018 polypeptides or antagonists,administered by subcutaneous injection one or more times per week. If anantibody against an IMX97018 polypeptide is used as the IMX97018polypeptide antagonist, a preferred dose range is 0.1 to 20 mg/kg, andmore preferably is 1-10 mg/kg. Another preferred dose range for ananti-IMX97018 polypeptide antibody is 0.75 to 7.5 mg/kg of body weight.Humanized antibodies are preferred, that is, antibodies in which onlythe antigen-binding portion of the antibody molecule is derived from anon-human source. Such antibodies can be injected or administeredintravenously.

Formulations. Compositions comprising an effective amount of an IMX97018polypeptide of the present invention (from whatever source derived,including without limitation from recombinant and non-recombinant,sources), in combination with other components such as a physiologicallyacceptable diluent, carrier, or excipient, are provided herein. The term“Pharmaceutically acceptable” means a non-toxic material that does notinterfere with the effectiveness of the biological activity of theactive ingredient(s). Formulations suitable for administration includeaqueous and non-aqueous sterile injection solutions which can containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions which can include suspending agents orthickening agents. The polypeptides can be formulated according to knownmethods used to prepare pharmaceutically useful compositions. They canbe combined in admixture, either as the sole active material or withother known active materials suitable for a given indication, withpharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate,and phosphate buffered solutions), preservatives (e.g., thimerosal,benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable formulations for pharmaceutical compositions includethose described in Remington's Pharmaceutical Sciences, 16th ed. 1980,Mack Publishing Company, Easton, Pa. In addition, such compositions canbe complexed with polyethylene glycol (PEG), metal ions, or incorporatedinto polymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, dextran, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts or spheroblasts. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871;U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No.4,737,323. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance, and are thus chosen according to the intended application, sothat the characteristics of the carrier will depend on the selectedroute of administration. In one preferred embodiment of the invention,sustained-release forms of IMX97018 polypeptides are used.Sustained-release forms suitable for use in the disclosed methodsinclude, but are not limited to, IMX97018 polypeptides that areencapsulated in a slowly-dissolving biocompatible polymer (such as thealginate microparticles described in U.S. Pat. No. 6,036,978), admixedwith such a polymer (including topically applied hydrogels), and orencased in a biocompatible semi-permeable implant.

Combinations of Therapeutic Compounds. AN IMX97018 polypeptide of thepresent invention may be active in multimers (e.g., heterodimers orhomodimers) or complexes with itself or other polypeptides. As a result,pharmaceutical compositions of the invention may comprise a polypeptideof the invention in such multimeric or complexed form. Thepharmaceutical composition of the invention may be in the form of acomplex of the polypeptide(s) of present invention along withpolypeptide or peptide antigens. The invention further includes theadministration of IMX97018 polypeptides or antagonists concurrently withone or more other drugs that are administered to the same patient incombination with the IMX97018 polypeptides or antagonists, each drugbeing administered according to a regimen suitable for that medicament.“Concurrent administration” encompasses simultaneous or sequentialtreatment with the components of the combination, as well as regimens inwhich the drugs are alternated, or wherein one component is administeredlong-term and the other(s) are administered intermittently. Componentscan be administered in the same or in separate compositions, and by thesame or different routes of administration. Examples of components thatcan be administered concurrently with the pharmaceutical compositions ofthe invention are: cytokines, lymphokines, or other hematopoieticfactors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-17,IL-18, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cellfactor, and erythropoietin, or inhibitors or antagonists of any of thesefactors. The pharmaceutical composition can further contain other agentswhich either enhance the activity of the polypeptide or compliment itsactivity or use in treatment. Such additional factors and/or agents maybe included in the pharmaceutical composition to produce a synergisticeffect with polypeptide of the invention, or to minimize side effects.Conversely, an IMX97018 polypeptide or antagonist of the presentinvention may be included in formulations of the particular cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent to minimize side effects of thecytokine, lymphokine, other hematopoietic factor, thrombolytic oranti-thrombotic factor, or anti-inflammatory agent. Additional examplesof drugs to be administered concurrently include but are not limited toantivirals, antibiotics, analgesics, corticosteroids, antagonists ofinflammatory cytokines, non-steroidal anti-inflammatories,pentoxifylline, thalidomide, and disease-modifying antirheumatic drugs(DMARDs) such as azathioprine, cyclophosphamide, cyclosporine,hydroxychloroquine sulfate, methotrexate, leflunomide, minocycline,penicillamine, sulfasalazine and gold compounds such as oral gold, goldsodium thiomalate, and aurothioglucose. Additionally, IMX97018polypeptides or antagonists can be combined with a second IMX97018polypeptide/antagonist, including an antibody against an IMX97018polypeptide, or an IMX97018 polypeptide-derived peptide that acts as acompetitive inhibitor of a native IMX97018 polypeptide.

Routes of Administration. Any efficacious route of administration can beused to therapeutically administer IMX97018 polypeptides or antagoniststhereof, including those compositions comprising nucleic acids.Parenteral administration includes injection, for example, viaintra-articular, intravenous, intramuscular, intralesional,intraperitoneal or subcutaneous routes by bolus injection or bycontinuous infusion, and also includes localized administration, e.g.,at a site of disease or injury. Other suitable means of administrationinclude sustained release from implants; aerosol inhalation and/orinsufflation; eyedrops; vaginal or rectal suppositories; buccalpreparations; oral preparations, including pills, syrups, lozenges, icecreams, or chewing gum; and topical preparations such as lotions, gels,sprays, ointments or other suitable techniques. Alternatively,polypeptideaceous IMX97018 polypeptides or antagonists may beadministered by implanting cultured cells that express the polypeptide,for example, by implanting cells that express IMX97018 polypeptides orantagonists. Cells may also be cultured ex vivo in the presence ofpolypeptides of the present invention in order to modulate cellproliferation or to produce a desired effect on or activity in suchcells. Treated cells can then be introduced in vivo for therapeuticpurposes. The polypeptide of the instant invention may also beadministered by the method of protein transduction. In this method, theIMX97018 polypeptide is covalently linked to a protein-transductiondomain (PTD) such as, but not limited to, TAT, Antp, or VP22 (Schwarzeet al., 2000, Cell Biology 10: 290-295). The PTD-linked peptides canthen be transduced into cells by adding the peptides to tissue-culturemedia containing the cells (Schwarze et al., 1999, Science 285:1569;Lindgren et al., 2000, TiPS 21: 99; Derossi et al., 1998, Cell Biology8: 84; WO 00/34308; WO 99/29721; and WO 99/10376). In anotherembodiment, the patient's own cells are induced to produce IMX97018polypeptides or antagonists by transfection in vivo or ex vivo with aDNA that encodes IMX97018 polypeptides or antagonists. This DNA can beintroduced into the patient's cells, for example, by injecting naked DNAor liposome-encapsulated DNA that encodes IMX97018 polypeptides orantagonists, or by other means of transfection. Nucleic acids of theinvention can also be administered to patients by other known methodsfor introduction of nucleic acid into a cell or organism (including,without limitation, in the form of viral vectors or naked DNA). WhenIMX97018 polypeptides or antagonists are administered in combinationwith one or more other biologically active compounds, these can beadministered by the same or by different routes, and can be administeredsimultaneously, separately or sequentially.

Oral Administration. When a therapeutically effective amount ofpolypeptide of the present invention is administered orally, polypeptideof the present invention will be in the form of a tablet, capsule,powder, solution or elixir. When administered in tablet form, thepharmaceutical composition of the invention can additionally contain asolid carrier such as a gelatin or an adjuvant. The tablet, capsule, andpowder contain from about 5 to 95% polypeptide of the present invention,and preferably from about 25 to 90% polypeptide of the presentinvention. When administered in liquid form, a liquid carrier such aswater, petroleum, oils of animal or plant origin such as peanut oil,mineral oil, soybean oil, or sesame oil, or synthetic oils can be added.The liquid form of the pharmaceutical composition can further containphysiological saline solution, dextrose or other saccharide solution, orglycols such as ethylene glycol, propylene glycol or polyethyleneglycol. When administered in liquid form, the pharmaceutical compositioncontains from about 0.5 to 90% by weight of polypeptide of the presentinvention, and preferably from about 1 to 50% polypeptide of the presentinvention.

Intravenous Administration. When a therapeutically effective amount ofpolypeptide of the present invention is administered by intravenous,cutaneous or subcutaneous injection, polypeptide of the presentinvention will be in the form of a pyrogen-free, parenterally acceptableaqueous solution. The preparation of such parenterally acceptablepolypeptide solutions, having due regard to pH, isotonicity, stability,and the like, is within the skill in the art. A preferred pharmaceuticalcomposition for intravenous, cutaneous, or subcutaneous injection shouldcontain, in addition to polypeptide of the present invention, anisotonic vehicle such as Sodium Chloride Injection, Ringer's Injection,Dextrose Injection, Dextrose and Sodium Chloride Injection, LactatedRinger's Injection, or other vehicle as known in the art. Thepharmaceutical composition of the present invention can also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art. The duration of intravenous therapyusing the pharmaceutical composition of the present invention will vary,depending on the severity of the disease being treated and the conditionand potential idiosyncratic response of each individual patient. It iscontemplated that the duration of each application of the polypeptide ofthe present invention will be in the range of 12 to 24 hours ofcontinuous intravenous administration. Ultimately the attendingphysician will decide on the appropriate duration of intravenous therapyusing the pharmaceutical composition of the present invention.

Tissue Administration. For compositions of the present invention whichare useful for neural tissue disorders, the therapeutic method includesadministering the composition topically, systematically, or locally asan implant or device. When administered, the therapeutic composition foruse in this invention is, of course, in a pyrogen-free, physiologicallyacceptable form. Further, the composition can desirably be encapsulatedor injected in a viscous form for delivery to the site of tissue damage.Topical administration may be suitable for wound healing and tissuerepair. Therapeutically useful agents other than a polypeptide of theinvention or antagonist thereof which may also optionally be included inthe composition as described above, can alternatively or additionally,be administered simultaneously or sequentially with the composition inthe methods of the invention. The composition can include a matrixcapable of delivering the polypeptide- or antagonist-containingcomposition to the site of tissue damage, providing a structure for thedeveloping tissue and optimally capable of being resorbed into the body.Such matrices can be formed of materials presently in use for otherimplanted medical applications. The choice of matrix material is basedon biocompatibility, biodegradability, mechanical properties, cosmeticappearance and interface properties. The particular application of thecompositions will define the appropriate formulation. Potential matricesfor the compositions can be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure polypeptides orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sintered hydroxapatite,bioglass, aluminates, or other ceramics Matrices can be comprised ofcombinations of any of the above mentioned types of material, such aspolylactic acid and hydroxyapatite or collagen and tricalciumphosphate.The bioceramics can be altered in composition, such as incalcium-aluminate-phosphate and processing to alter pore size, particlesize, particle shape, and biodegradability. Presently preferred is a50:50 (mole weight) copolymer of lactic acid and glycolic acid in theform of porous particles having diameters ranging from 150 to 800microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the polypeptide compositions from disassociating fromthe matrix. A preferred family of sequestering agents is cellulosicmaterials such as alkylcelluloses (including hydroxyalkylcelluloses),including methylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethyl-cellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorption of the polypeptide or antagonist fromthe polymer matrix and to provide appropriate handling of thecomposition, yet not so much that the progenitor cells are preventedfrom infiltrating the matrix, thereby providing the polypeptide orantagonist the opportunity to assist the activity of the progenitorcells. In further compositions, polypeptides of the invention orantagonists thereof may be combined with other agents beneficial to thetreatment of the wound or tissue in question. These agents includevarious growth factors such as epidermal growth factor (EGF), plateletderived growth factor (PDGF), transforming growth factors (TGF-alpha andTGF-beta), and insulin-like growth factor (IGF). The dosage regimen of apolypeptide-containing pharmaceutical composition to be used in tissueregeneration will be determined by the attending physician consideringvarious factors which modify the action of the polypeptides, e.g.,amount of tissue weight desired to be formed, the site of damage, thecondition of the damaged tissue, the size of a wound, type of damagedtissue, the patient's age, sex, and diet, the severity of any infection,time of administration and other clinical factors. The dosage can varywith the type of matrix used in the reconstitution and with inclusion ofother polypeptides in the pharmaceutical composition. For example, theaddition of other known growth factors, such as IGF I (insulin likegrowth factor I), to the final composition, may also effect the dosage.Progress can be monitored by periodic assessment of tissue growth and/orrepair, for example, X-rays, histomorphometric determinations, andtetracycline labeling.

Veterinary Uses. In addition to human patients, IMX97018 polypeptidesand antagonists are useful in the treatment of disease conditions innon-human animals, such as pets (dogs, cats, birds, primates, etc.),domestic farm animals (horses cattle, sheep, pigs, birds, etc.),thoroughbred horses, or any animal that suffers from anIMX97018-mediated condition. In such instances, an appropriate dose canbe determined according to the animal's body weight. For example, a doseof 0.2-1 mg/kg may be used. Alternatively, the dose is determinedaccording to the animal's surface area, an exemplary dose ranging from0.1-20 mg/m², or more preferably, from 5-12 mg/m². For small animals,such as dogs or cats, a suitable dose is 0.4 mg/kg. In a preferredembodiment, IMX97018 polypeptides or antagonists (preferably constructedfrom genes derived from the same species as the patient), isadministered by injection or other suitable route one or more times perweek until the animal's condition is improved, or it can be administeredindefinitely.

Manufacture of Medicaments. The present invention also relates to theuse of IMX97018 polypeptides, fragments, and variants; nucleic acidsencoding the IMX97018 family polypeptides, fragments, and variants;agonists or antagonists of the IMX97018 polypeptides such as antibodies;IMX97018 polypeptide binding partners; complexes formed from theIMX97018 family polypeptides, fragments, variants, and binding partners,etc, in the manufacture of a medicament for the prevention ortherapeutic treatment of each medical disorder disclosed herein.

EXAMPLES

The following examples are intended to illustrate particular embodimentsand not to limit the scope of the invention.

Example 1 Identification of IMX97018, a New Human Ataxin-1-LikePolypeptide

A partial cDNA sequence was identified in a cDNA library prepared fromhuman dendritic cells (described in U.S. Pat. No. 6,017,729, issued Jan.25, 2000). That cDNA was used to identify a human chromosome 16 genomiccontig (AC009127.8) that does include the entire IMX97018 codingsequence, shown as SEQ ID NO:1. The entire IMX97018 coding sequence wasdetermined based on comparisons of human ataxin-1 polypeptide with allpossible reading frames of AC009127, using the GCG program TFASTA.Nucleotides 1 through 2067 of SEQ ID NO:1 encode the IMX97018polypeptide, the amino acid sequence of which is shown as SEQ ID NO:2.The approximate position of the single exon containing IMX97018 codingsequence in the AC009127.8 contig is shown in the table below, alongwith its location relative to SEQ ID NO:1; note that the coding sequenceis in the inverse orientation with respect to the contig, and that the5′ and 3′ untranslated regions may extend further along the contigsequence beyond those portions that correspond to SEQ ID NO:1, asindicated by the parentheses around the AC009127.8 positions in thetable.

Corresponding position of IMX97018 gene exon in human contig AC009127.8and in SEQ ID NO:1: Position in AC009127.8 Position in SEQ ID NO: 1 Exon1 (162604)-(160538) 1-2067

The human genomic region corresponding to the AC009127.8 contig, 16q22,also includes the genetic map location for SCA4. SCA4 was mapped byFlanigan et al. (1996, Am J Hum Genet 59: 392-399) to a 6 centiMorganregion around the D16S397 marker on 16q22.1, and bounded on the distalend by D16S512 at 16q22.3. The AC009127.8 contig maps between D16S397and D16S512 within 16q22.3. Therefore, the ataxin-1-like IMX97018polypeptide is considered to be a strong candidate for the product ofthe SCA4 gene.

Additional variations of IMX97018 polypeptides are provided as naturallyoccurring genomic variants of the IMX97018 sequences disclosed herein;such variations may be incorporated into an IMX97018 polypeptide ornucleic acid individually or in any combination. As one example, the GGCcodon encoding the Gly residue at position 428 of SEQ ID NO:2 is presentas an AGC codon encoding a Ser residue in a naturally occurring variantof SEQ ID NO: 1, likely representing an allelic variation.

The amino acid sequence of IMX97018 (SEQ ID NO:2) was compared with theamino acid sequences of ataxin-1-like polypeptides—human (‘Hs’), mouse(‘Mm’), and rat (‘Rn’) ataxin-1 (SEQ ID NO:3-SEQ ID NO:5,respectively)—using the GCG “pretty” multiple sequence alignmentprogram, with amino acid similarity scoring matrix=blosum62, gapcreation penalty=8, and gap extension penalty=1. An alignment of thesesequences is shown in Table 1, and includes consensus residues which areidentical among all four of the amino acid sequences in the alignment.The capitalized residues in the alignment are those which match theconsensus residues. The numbering of amino acid residues in Table 1corresponds to the position of those residues in the IMX97018 amino acidsequence (SEQ ID NO:2). The AXH ataxin-1 and HMG-box-containingconserved domain sequence (SEQ ID NO:6) has been optimally aligned withthe Table 1 amino acid sequences, and residues within the AXH sequencethat match the Table 1 consensus residues are capitalized.

Amino acid substitutions and other alterations (deletions, insertions,etc.) to IMX97018 amino acid sequences (e.g. SEQ ID NO:2) are predictedto be more likely to alter or disrupt IMX97018 polypeptide activities ifthey result in changes to the capitalized residues of the amino acidsequences as shown in Table 1, and particularly if those changes do notsubstitute an amino acid of similar structure (such as substitution ofany one of the aliphatic residues—Ala, Gly, Leu, Ile, or Val—for anotheraliphatic residue), or a residue present in ataxin-1 polypeptides or theAXH domain at that conserved position. Conversely, if a change is madeto an IMX97018 amino acid sequence resulting in a substitution of theresidue at that position in the alignment from one of the other Table 1ataxin-1 polypeptide or AXH domain sequences, it is less likely thatsuch an alteration will affect the function of the altered IMX97018polypeptide. For example, the consensus residue at position 504 in Table1 is lysine, and the AXH domain has an aspartate at that position.Substitution of aspartate or the chemically similar glutamate for lysineat that position is considered less likely to alter the function of thepolypeptide than substitution of tryptophan or tyrosine etc. Embodimentsof the invention include IMX97018 polypeptides and fragments of IMX97018polypeptides, comprising altered amino acid sequences. Altered IMX97018polypeptide sequences share at least 30%, or more preferably at least40%, or more preferably at least 50%, or more preferably at least 55%,or more preferably at least 60%, or more preferably at least 65%, ormore preferably at least 70%, or more preferably at least 75%, or morepreferably at least 80%, or more preferably at least 85%, or morepreferably at least 90%, or more preferably at least 95%, or morepreferably at least 97.5%, or more preferably at least 99%, or mostpreferably at least 99.5% amino acid identity with one or more of theamino acid sequences shown in Table 1. When IMX97018 polypeptidevariants according to the invention, such as allelic variants orIMX97018 polypeptides having deliberately engineered modifications, areanalyzed using the GeneFold algorithm (Tripos Inc., St. Louis, Mo., seetripos.com/admin/LitCtr/genefold app.pdf), the top five scoring templatestructures will be ataxin-1 or AXH-domain-containing polypeptides. Thescore for these top five hits, using any of the three types of scorereported by GeneFold (sequence only, sequence plus local conformationpreferences plus burial terms, or sequence plus local conformationpreferences plus burial terms plus secondary structure) preferably willbe at least 500, more preferably at least 750, and most preferably999.9. TABLE 1 Alignment of IMX97018 amino acid sequence with those ofataxin-1 polypeptides Protein (SEQ ID NO) 1             50 IMX97018(2)MKpvhERsqE CLPPKKRdlP vTSedmgrtt scstnhtpss daseWsrgvv Hs ATX1(3)MKsnqERsnE CLPPKKReiP aTSrsseeka ptlpsdnhrv egtaWlpg.n Mm ATX1(4)MKsnqERtnE CLPPKKReiP aTSrpseeka talpsdnhcv egvaWlps.t Rn ATX1(5)MKsnqERsnE CLPPKKReiP aTSrpseeka talpsdnhcv egvaWlps.t consensusMK---ER--E CLPPKIKR--P -TS------- ---------- ----W-----51             98 IMX97018(2) vagqsqaGaR vslgGdgaEa itGLtvdqyGm.LyKvavpp atfSPtglP. Hs ATX1(3) pggrghgGgR hgpaGtsvE. .lGL . . . qqGigLhKalstg ldySPpsaPr Mm ATX1(4) pgirghgGgR hgsaGtsgE. .hGL . . . qGmgLlKalsag ldySPpsaPr Rn ATX1(5) pgsrghgGgR hgpaGtsgE. .hGL . . . qGmgLhKalsag ldySPpsaPr consensus -------G-R ----G---E- --GL-----G--L-K----- ---SP---P- 99             147 IMX97018(2) SVvnmspLPptfnvassliq hpGihypPlh YAqLpsTslQ FIG.SpYSlp Hs ATX1(3) SVpvattLPaayatpqp . . . GtpvsPvq YAhLphT.fQ FIGsSqYSgt Mm ATX1(4) SVptantLPtvypppqs . . . GtpvsPvq YAhLshT.fQ FIGsSqYSgp Rn ATX1(5) SVptantLPtvypppqs . . . GtpvsPvq YAhLshT.fQ FIGsSqYSgp consensus SV-----LP---G----P-- YA-L--T--Q FIG-S-YS-- 148            190 IMX97018(2)YAvppnFlPS pLlsPsaNla TShlphfvpy AsllAeGATp PpQ

Hs ATX1(3) YA . . . sFiPS qLipPtaNpv TS . . . AvasAaGATt PsQrsqleay MmATX1(4) YA . . . gFiPS qLisPsgNpv TS . . . AvasAaGATt PsQrsqleay RnATX1(5) YA . . . gFiPS qLisPpgNpv TS . . . AvasAaGATt PsQrsqleayconsensus YA----F-PS -L--P--N-- TS-------- A---A-GAT- P-Q-------           191 197 IMX97018(2)

apspahs Hs ATX1(3) stllanmgsl sqtpghkaeq qqqqqqqqqq qhqhqqqqqqqqqqqqqqqh Mm ATX1(4) stllanmgsl sqapghkve . . . pppqqh Rn ATX1(5)stllanmgsl sqapghkve . . . pppqqh consensus ---------- -------------------- ---------- ----------              

198            236 IMX97018(2) fnkApsatsP sgqlPh . . . HsStqPl d . . .lapgr mPiyyqmsrl Hs ATX1(3) lsrApglitP gs.pPpaqqn qyvHiSssPq ntgrtasppaiPvhlhphqt Mm ATX1(4) lsrAaglvnP gsppPptqqn qyiHiSssPq ssgratspppiPvhlhphqt Rn ATX1(5) lgrAaglvnP gs.pPptqqn qyiHiSssPq ssgrats.ppiPvhlhphqt consensus ---A-----P ----P----- ---H-S--P- -----------P-------- 237            270 IMX97018(2) pagyTLhetP p . . . agaspvltPqEs . . . Q sAleAaaang Hs ATX1(3) miphTLtlgP psqvvmqyadsgshfvPrEa tkkaessrlQ qAiqAkevln Mm ATX1(4) miphTLtlgP ssqvvvqysdagghfvPrEs tkkaessrlQ qAmqAkevln Rn ATX1(5) miphTLtlgP ssqvvvqysdagghfvPrEs tkkaessrlQ qAmqAkevln consensus ----TL---P ----------------P-E- ---------Q -A--A----- 271            313 IMX97018(2)GqrpreRnlv rreSe . . . aLd spnsKg . . . EgqglVpVv ecvvDgqlfS Hs ATX1(3)GemeksRryg apsSadlgLg kaggKsvphp yEsrhvV.Vh pspsD . . . yS Mm ATX1(4)GemeksRryg assSvelsLg kassKsvphp yEsrhvV.Vh pspaD . . . yS Rn ATX1(5)GemeksRryg assSvelsLg ktssKsvphp yEsrhvV.Vh pspaD . . . yS consensusG-----R--- ---S----L- ----K---- -E----V-V- ----D----S 314            361IMX97018(2) gsqtp . . . Rve VaapahrgTP dtDLEvQrvv galasqdyrv vaaqRkeePSHs ATX1(3) srdpsgvRas VmvlpnsnTP aaDLEvQq . . . athReasPS Mm ATX1(4)srdtsgvRgs VmvlpnssTP saDLEaQq . . . tthReasPS Rn ATX1(5) srdtsgvRgsVmvlpnssTP saDLEtQq . . . athReasPS consensus -------R-- V-------TP--DLE-Q--- ---------- ----R---PS 362            398 IMX97018(2) pLN . .. Lsh htPdHqg . . . egrg SArnPaeLae ksqArgFYpq Hs ATX1(3) tLNdksgLhlgkPgHrsyal sphtviqtth SAseP . . . Lpv glpAtaFYag Mm ATX1(4) tLNdksgLaprkPgHrsyal sphtviqtth SAseP . . . Lpv glpAtaFYag Rn ATX1(5) tLNdksgLhlgkPgHrsyal sphtviqtth SAseP . . . Lpv glpAtaFYag consensus -LN----L----P-H----- ---------- SA--P--L-- ---A--FY- 399            434IMX97018(2) shQePV . . . kh rplpkAmvvA ng . . . nLVpt GtdsgLlPVG S . . .Hs ATX1(3) t.QpPVigyl sgqqqAityA gslpqhLVip GtqplLiPVG Stdmeasgaa MmATX1(4) t.QpPVigyl sgqqqAityA gglpqhLVip GnqplALiPVG Spdmdmpgaa RnATX1(5) a.QpPVigyl ssqqqAityA gglpqhLVip GtqplLiPVG Spdmdtpgaa consensus--Q-PV---- -----A---A ------LV-- G----L-PVG S--------- Self-ass'n          λλλλλλλλλλλλλλλλλλλλλλλλλλλ 435            451 IMX97018(2)eilVaSSld . . . VQAratfP Hs ATX1(3) paiVtSSpqf aavphtfvtt alpksenfnpealvtqaayp amVQAqihlP Mm ATX1(4) saiVtSSpqf aavphtfvtt alpksenfhpealvtqasyp amVQAqihlP Rn ATX1(5) saiVtSSpqf aavphtfvtt alpksenfnpealvtqaayp amVQAqihlP consensus ---V-SS--- ---------- -------------------- --VQA----P Self-ass'nλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλλ RNA-bindng

452            500 IMX97018(2) dkeptppPit ss.hLPshFM KGaIIQLAtGELKrVEDLqT qDFvrSAEvS Hs ATX1(3) vvqsvasPaa apptLPpyFM KGsIIQLAnGELKkVEDLkT eDFiqSAEiS Mm ATX1(4) vvqsvasPtt asptLPpyFM KGsIIQLAnGELKkVEDLkT eDFiqSAEiS Rn ATX1(5) vvqsvasPaa asptLPpyFM KGsIIQLAnGELKkVEDLkT eDFiqSAEiS AXH (6) tvPhcFM KGtr1cLAnG snKkVEDLrT eDFirSAgcSconsensus -------P-- ----LP--FM KG-IIQLA-G ELK-VEDL-T -DF--SAE-SSelf-ass'n

RNA-bindng

501            550 IMX97018(2) ggLKIdSSTV vdIqeSqwPG fvmlhFvVGEqqskVSiEVp pEhPFFVyGQ Hs ATX1(3) ndLKIdSSTV erIedShsPG vaviqFaVGEhraqVSvEVl vEyPFFVfGQ Mm ATX1(4) ndLKIhSSTV erIeeShsPG vaviqFaVGEhraqVSvEVl vEyPFFVfGQ Rn ATX1(5) ndLKIdSSTV erIedShsPG vaviqFaVGEhraqVSvEVl vEyPFFVfGQ AXH (6) ndedlqmSTV krIgsSglPs vvtltFdpGvedalltvEcq vEhPFFVkGk consensus --LKI-SSTV --I--S--PG-----F-VGE----VS-EV- -E-PFFV-GQ RNA-bindng

551            600 IMX97018(2) GWSSCsPgRT tQLFsLPChr LqVGDVCISisLqsLnsnSV sqascapPsq Hs ATX1(3) GWSSCcPeRT sQLFdLPCsk LsVGDVCISltLknLkngSV kkgqpvdPas Mm ATX1(4) GWSSCcPeRT sQLFdLPCsk LsVGDVCISltLknLkngSV kkgqpvdPas Rn ATX1(5) GWSSCcPeRT sQLFdLPCsk LsVGDVCISltLknLkngSV kkgqpvdPas AXH (6) GWSSCyPslT vQLygLPCce LqVGDVClSl thnconsensus GWSSC-P-RT -QLF-LPC-- L-VGDVCIS- -L--L---SV -------P--RNA-bindng

601            629 IMX97018(2) . . . LgppReR . . . pErtv.lGSr elcdseGksq. . . P Ag Hs ATX1(3) vllkhskadg LagsRhRyae qEnginqGSa qmlsenGelkfpekmglP Aa Mm ATX1(4) vllkqvktds LagsRhRyae qEnginqGSa qvlsenGelkfpekiglPAa Rn ATX1(5) allkhaktds LagsRhRyae qEnginqGSa qvlsenGelkfpekiglPAa consensus ---------- L---R-R--- -E-----GS- ------G----------PA- RNA-bindng

630            678 IMX97018(2) egsrvvEPSq Pesgaqa.cW pAPsfqrysmqgeEaraaLl rPSfIPQEVK Hs ATX1(3) pfltkiEPSk Paatrk.rrW sAPesrkleksedEppltLp kPSlIPQEVK Mm ATX1(4) pflskiEPSk PtatrkrrrW sAPetrkleksedEppltLp kPSlIIPQEVK Rn ATX1(5) pfltkiEPSk Ptatrk.rrW sAPetrkleksedEppltLp kPSlIPQEVK consensus ------EPS- P--------W -AP----------E----L- -PS-IPQEVK RNA-bindng

679  689 IMX97018(2) lsEGRSNaG K Hs ATX1(3) icIEGRSNvG K Mm ATX1(4)icIEGRSNvG K Rn ATX1(5) icIEGRSNvG K consensus --IEGRSN-G K

Expression of polynucleotides encoding IMX97018 polypeptides. An arrayof polynucleotides, including polynucleotide probes specific forIMX97018-encoding sequences, was contacted with human RNA samplesprepared from a variety of tissues and cell types. Low levels ofexpression of IMX97018-encoding sequences were detected in all samplestested.

Example 2 Monoclonal Antibodies that Bind Polypeptides of the Invention

This example illustrates a method for preparing monoclonal antibodiesthat bind IMX97018 polypeptides. Other conventional techniques may beused, such as those described in U.S. Pat. No. 4,411,993. Suitableimmunogens that may be employed in generating such antibodies include,but are not limited to, purified IMX97018 polypeptide, an immunogenicfragment thereof, and cells expressing high levels of IMX97018polypeptide or an immunogenic fragment thereof. DNA encoding an IMX97018polypeptide can also be used as an immunogen, for example, as reviewedby Pardoll and Beckerleg in Immunity 3: 165, 1995.

Rodents (BALB/c mice or Lewis rats, for example) are immunized withIMX97018 polypeptide immunogen emulsified in an adjuvant (such ascomplete or incomplete Freund's adjuvant, alum, or another adjuvant,such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.)), and injected inamounts ranging from 10-100 micrograms subcutaneously orintraperitoneally. DNA may be given intradermally (Raz et al., 1994,Proc. Natl. Acad. Sci. USA 91: 9519) or intamuscularly (Wang et al.,1993, Proc. Natl. Acad. Sci. USA 90: 4156); saline has been found to bea suitable diluent for DNA-based antigens. Ten days to three weeks dayslater, the immunized animals are boosted with additional immunogen andperiodically boosted thereafter on a weekly, biweekly or every thirdweek immunization schedule.

Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision to test for IMX97018 polypeptide antibodies bydot-blot assay, ELISA (enzyme-linked immunosorbent assay),immunoprecipitation, or other suitable assays, such as FACS analysis ofinhibition of binding of IMX97018 polypeptide to an IMX97018 polypeptidebinding partner. Following detection of an appropriate antibody titer,positive animals are provided one last intravenous injection of IMX97018polypeptide in saline. Three to four days later, the animals aresacrificed, and spleen cells are harvested and fused to a murine myelomacell line, e.g., NS1 or preferably P3X63Ag8.653 (ATCC CRL-1580). Thesecell fusions generate hybridoma cells, which are plated in multiplemicrotiter plates in a HAT (hypoxanthine, aminopterin and thymidine)selective medium to inhibit proliferation of non-fused cells, myelomahybrids, and spleen cell hybrids.

The hybridoma cells may be screened by ELISA for reactivity againstpurified IMX97018 polypeptide by adaptations of the techniques disclosedin Engvall et al., (Immunochem. 8: 871, 1971) and in U.S. Pat. No.4,703,004. A preferred screening technique is the antibody capturetechnique described in Beckmann et al., (J. Immunol. 144: 4212, 1990).Positive hybridoma cells can be injected intraperitoneally intosyngeneic rodents to produce ascites containing high concentrations (forexample, greater than 1 milligram per milliliter) of anti-IMX97018polypeptide monoclonal antibodies. Alternatively, hybridoma cells can begrown in vitro in flasks or roller bottles by various techniques.Monoclonal antibodies can be purified by ammonium sulfate precipitation,followed by gel exclusion chromatography. Alternatively, affinitychromatography based upon binding of antibody to protein A or protein Gcan also be used, as can affinity chromatography based upon binding toIMX97018 polypeptide.

Example 3 Antisense Inhibition of IMX97018 Nucleic Acid Expression

In accordance with the present invention, a series of oligonucleotidesare designed to target different regions of the IMX97018 mRNA molecule,using the nucleotide sequence of SEQ ID NO: 1 as the basis for thedesign of the oligonucleotides. The oligonucleotides are selected to beapproximately 10, 12, 15, 18, or more preferably 20 nucleotide residuesin length, and to have a predicted hybridization temperature that is atleast 37 degrees C. Preferably, the oligonucleotides are selected sothat some will hybridize toward the 5′ region of the mRNA molecule,others will hybridize to the coding region, and still others willhybridize to the 3′ region of the mRNA molecule. Methods such as thoseof Gray and Clark (U.S. Pat. Nos. 5,856,103 and 6,183,966) can be usedto select oligonucleotides that form the most stable hybrid structureswith target sequences, as such oligonucleotides are desirable for use asantisense inhibitors.

The oligonucleotides may be oligodeoxynucleotides, with phosphorothioatebackbones (internucleoside linkages) throughout, or may have a varietyof different types of internucleoside linkages. Generally, methods forthe preparation, purification, and use of a variety of chemicallymodified oligonucleotides are described in U.S. Pat. No. 5,948,680. Asspecific examples, the following types of nucleoside phosphoramiditesmay be used in oligonucleotide synthesis: deoxy and 2′-alkoxy amidites;2′-fluoro amidites such as 2′-fluorodeoxyadenosine amidites,2′-fluorodeoxyguanosine, 2′-fluorouridine, and 2′-fluorodeoxycytidine;2′-O-(2-methoxyethyl)-modified amidites such as2,2′-anhydro[1-(beta-D-arabino-furanosyl)-5-methyluridine],2′-O-methoxyethyl-5-methyluridine,2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine,3′-O-acetyl-2′-O-methoxy-ethyl-5′-O-dimethoxytrityl-5-methyluridine,3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine,2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine,N4-benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine, andN4-benzoyl-2′-O-methoxyethyl-5′-O-di-methoxytrityl-5-methylcytidine-3′-amidite;2′-O-(aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites such as2′-(dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-butyl-diphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenyl-silyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxy-ethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, and5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphor-amidite];and 2′-(aminooxyethoxy) nucleoside amidites such asN2-isobutyryl-6-O-diphenyl-carbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diiso-propylphosphoramidite].

Modified oligonucleosides may also be used in oligonucleotide synthesis,for example methylenemethylimino-linked oligonucleosides, also calledMMI-linked oligonucleosides; methylene-dimethylhydrazo-linkedoligonucleosides, also called MDH-linked oligonucleosides;methylene-carbonylamino-linked oligonucleosides, also calledamide-3-linked oligonucleosides; and methylene-aminocarbonyl-linkedoligonucleosides, also called amide-4-linked oligonucleosides, as wellas mixed backbone compounds having, for instance, alternating MMI andP═O or P═S linkages, which are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289. Formacetal-and thioformacetal-linked oligonucleosides may also be used and areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564; andethylene oxide linked oligonucleosides may also be used and are preparedas described in U.S. Pat. No. 5,223,618. Peptide nucleic acids (PNAs)may be used as in the same manner as the oligonucleotides describedabove, and are prepared in accordance with any of the various proceduresreferred to in Peptide Nucleic Acids (PNA): Synthesis, Properties andPotential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23;and U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262.

Chimeric oligonucleotides, oligonucleosides, or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”. Someexamples of different types of chimeric oligonucleotides are:[2′-O-Me]-[2′-deoxy]-[2′-O-Me] chimeric phosphorothioateoligonucleotides,[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides, and[2′-O-(2-methoxy-ethyl)phosphodiester]-[2′deoxyphosphoro-thioate]-[2′-O-(2-methoxyethyl)phosphodiester] chimericoligonucleotides, all of which may be prepared according to U.S. Pat.No. 5,948,680. In one preferred embodiment, chimeric oligonucleotides(“gapmers”) 18 nucleotides in length are utilized, composed of a central“gap” region consisting of ten 2′-deoxynucleotides, which is flanked onboth sides (5′ and 3′ directions) by four-nucleotide “wings”. The wingsare composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. Cytidine residues in the 2′-MOE wingsare 5-methylcytidines. Other chimeric oligonucleotides, chimericoligonucleosides, and mixed chimeric oligonucleotides/oligonucleosidesare synthesized according to U.S. Pat. No. 5,623,065.

Oligonucleotides are preferably synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format. Theconcentration of oligonucleotide in each well is assessed by dilution ofsamples and UV absorption spectroscopy. The full-length integrity of theindividual products is evaluated by capillary electrophoresis, and baseand backbone composition is confirmed by mass analysis of the compoundsutilizing electrospray-mass spectroscopy.

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Cells areroutinely maintained for up to 10 passages as recommended by thesupplier. When cells reached 80% to 90% confluency, they are treatedwith oligonucleotide. For cells grown in 96-well plates, wells arewashed once with 200 microliters OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 microliters of OPTI-MEM-1 containing 3.75g/mL LIPOFECTIN (Gibco BRL) and the desired oligonucleotide at a finalconcentration of 150 nM. After 4 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours afteroligonucleotide treatment. Preferably, the effect of several differentoligonucleotides should be tested simultaneously, where theoligonucleotides hybridize to different portions of the target nucleicacid molecules, in order to identify the oligonucleotides producing thegreatest degree of inhibition of expression of the target nucleic acid.

Antisense modulation of IMX97018 nucleic acid expression can be assayedin a variety of ways known in the art. For example, IMX97018 mRNA levelscan be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-timequantitative PCR is presently preferred. RNA analysis can be performedon total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation andNorthern blot analysis are taught in, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1996. Real-time quantitative(PCR) can be conveniently accomplished using the commercially availableABI PRISM 7700 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions. This fluorescence detection system allows high-throughputquantitation of PCR products. As opposed to standard PCR, in whichamplification products are quantitated after the PCR is completed,products in real-time quantitative PCR are quantitated as theyaccumulate. This is accomplished by including in the PCR reaction anoligonucleotide probe that anneals specifically between the forward andreverse PCR primers, and contains two fluorescent dyes. A reporter dye(e.g., JOE or FAM, obtained from either Operon Technologies Inc.,Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular (six-second) intervals bylaser optics built into the ABI PRISM 7700 Sequence Detection System. Ineach assay, a series of parallel reactions containing serial dilutionsof mRNA from untreated control samples generates a standard curve thatis used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples. Other methods of quantitativePCR analysis are also known in the art. IMX97018 protein levels can bequantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting), ELISA, orfluorescence-activated cell sorting (FACS). Antibodies directed toIMX97018 polypeptides can be prepared via conventional antibodygeneration methods such as those described herein. Immunoprecipitationmethods, Western blot (immunoblot) analysis, and enzyme-linkedimmunosorbent assays (ELISA) are standard in the art (see, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2,pp. 10.16.1-10.16.11, 10.8.1-10.8.21, and 11.2.1-11.2.22, John Wiley &Sons, Inc., 1991).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims. SequencesPresented in the Sequence Listing SEQ ID NO Type Description SEQ ID NO:1 Nucleotide IMX97018 coding sequence SEQ ID NO: 2 Amino acid IMX97018amino acid sequence SEQ ID NO: 3 Amino acid Homo sapiens ataxin-1polypeptide (SWISSPROT accession # P54253) SEQ ID NO: 4 Amino acid Musmusculus ataxin-1 polypeptide (SWISSPROT accession # P54254) SEQ ID NO:5 Amino acid Rattus norvegicus ataxin-1 polypeptide (GenBank accession #NP_036858) SEQ ID NO: 6 Amino acid AXH domain (ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml; smart00536)

1. An isolated antibody that binds to a polypeptide consisting of anamino acid sequence selected from the group consisting of: (a) the aminoacid sequence of SEQ ID NO:2; (b) an amino acid sequence selected fromthe group consisting of: amino acids 542 through 579 of SEQ ID NO:2, andamino acids 464 through 583 of SEQ ID NO:2; (c) an amino acid sequenceselected from the group consisting of: amino acids 465 through 499 ofSEQ ID NO:2, and amino acids 431 through 499 of SEQ ID NO:2; (d) anamino acid sequence selected from the group consisting of: amino acids465 through 590 of SEQ ID NO:2, and amino acids 444 through 640 of SEQID NO:2; (e) a fragment of the amino acid sequences of any of (a)-(d)having IMX97018 polypeptide self-association or RNA-binding activity;and (f) a fragment of the amino acid sequences of any of (a)-(d)comprising the AXH.
 2. The antibody of claim 1, wherein the antibody isan antagonistic antibody.
 3. The antibody of claims 1 or 2, wherein theantibody is a monoclonal antibody.
 4. A hybridoma capable of producingthe antibody of claim 3.